Zika RNA vaccines

ABSTRACT

Aspects of the disclosure describe compositions that comprise RNA polynucleotides having an open reading frame encoding one or more Zika virus antigens. Methods for preparing and using the compositions are also described.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/746,286, filed Jan. 19, 2018, entitled “INFECTIOUS DISEASE VACCINES”,which is a national stage filing under 35 U.S.C. § 371 of InternationalPatent Application Serial No. PCT/US2016/043348, filed Jul. 21, 2016,which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication No. 62/195,263, filed Jul. 21, 2015, U.S. provisionalapplication No. 62/241,699, filed Oct. 14, 2015, U.S. provisionalapplication No. 62/244,859, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,551, filed Oct. 28, 2015, and U.S. provisionalapplication No. 62/351,148, filed Jun. 16, 2016, each of which isincorporated by reference herein in its entirety. This application alsoclaims the benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication No. 62/244,855, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,527, filed Oct. 28, 2015, U.S. provisionalapplication No. 62/303,405, filed Mar. 4, 2016, and U.S. provisionalapplication No. 62/351,200, filed Jun. 16, 2016, each of which isincorporated by reference herein in its entirety. This application alsoclaims the benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication No. 62/199,204, filed Jul. 30, 2015, U.S. provisionalapplication No. 62/245,179, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,581, filed Oct. 28, 2015, and U.S. provisionalapplication No. 62/351,206, filed Jun. 16, 2016, each of which isincorporated by reference herein in its entirety. This application alsoclaims the benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication No. 62/245,233, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,660, filed Oct. 28, 2015, U.S. provisionalapplication No. 62/303,666, filed Mar. 4, 2016, U.S. provisionalapplication No. 62/351,244, filed Jun. 16, 2016, and U.S. provisionalapplication No. 62/357,806, filed Jul. 1, 2016, each of which isincorporated by reference herein in its entirety. This application alsoclaims the benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication No. 62/244,995, filed Oct. 22, 2015, U.S. provisionalapplication No. 62/247,644, filed Oct. 28, 2015, and U.S. provisionalapplication No. 62/351,267, filed Jun. 16, 2016, each of which isincorporated by reference herein in its entirety.

BACKGROUND OF INVENTION

Chikungunya virus (CHIKV) is a mosquito-borne virus belonging to theAlphavirus genus of the Togaviridae family that was first isolated in1953 in Tanzania, where the virus was endemic. Outbreaks occurrepeatedly in west, central, and southern Africa and have caused severalhuman epidemics in those areas since that time. The virus is passed tohumans by two species of mosquito of the genus Aedes: A. albopictus andA. aegypti. There are several Chikungunya genotypes: Indian Ocean,East/Central/South African (ECSA), Asian, West African, and Brazilian.

Presently, CHIKV is a re-emerging human pathogen that has nowestablished itself in Southeast Asia and has more recently spread toEurope. The Chikungunya virus (CHIKV) was introduced into Asia around1958, and sites of endemic transmission within Southeastern Asia,including the Indian Ocean, were observed through 1996. The CHIKVepidemic moved throughout Asia, reaching Europe and Africa in the early2000s, and was imported via travelers to North America and South Americafrom 2005 to 2007. Sporadic outbreaks are still occurring in severalcountries, such as Italy, inflicting naive populations. Singapore, forinstance, experienced two successive waves of Chikungunya virusoutbreaks in January and August 2008. Of the two strain lineages ofCHIKV, the African strain remains enzootic by cycling between mosquitoesand monkeys, but the Asian strain is transmitted directly betweenmosquitoes and humans. This cycle of transmission may have allowed thevirus to become more pathogenic as the reservoir host was eliminated.

In humans, CHIKV causes a debilitating disease characterized by fever,headache, nausea, vomiting, fatigue, rash, muscle pain and joint pain.Following the acute phase of the illness, patients develop severechronic symptoms lasting from several weeks to months, includingfatigue, incapacitating joint pain and polyarthritis.

The re-emergence of CHIKV has caused millions of cases throughoutcountries around the Indian Ocean and in Southeast Asia. Specifically,India, Indonesia, Maldives, Myanmar and Thailand have reported over 1.9million cases since 2005. Globally, human CHIKV epidemics from 2004-2011have resulted in 1.4-6.5 million reported cases, including a number ofdeaths. Thus, CHIKV remains a public threat that constitutes a majorpublic health problem with severe social and economic impact.

Despite significant morbidity and some cases of mortality associatedwith CHIKV infection and its growing prevalence and geographicdistribution, there is currently no licensed CHIKV vaccine or antiviralapproved for human use. Several potential CHIKV vaccine candidates havebeen tested in humans and animals with varying success.

Dengue virus (DENV) is a mosquito-borne (Aedes aegypti/Aedes albopictus)member of the family Flaviviridae (positive-sense, single-stranded RNAvirus). Dengue virus is a positive-sense RNA virus of the Flavivirusgenus of the Flaviviridae family, which also includes West Nile virus,Yellow Fever Virus, and Japanese Encephalitis virus. It is transmittedto humans through Stegomyia aegypti (formerly Aedes) mosquito vectorsand is mainly found in the tropical and semitropical areas of the world,where it is endemic in Asia, the Pacific region, Africa, Latin America,and the Caribbean. The incidence of infections has increased 30-foldover the last 50 years (WHO, Dengue: Guidelines for diagnosis,treatment, prevention, and control (2009)) and Dengue virus is thesecond most common tropical infectious disease worldwide after malaria.

There is no specific treatment for DENV infection, and control of DENVby vaccination has proved elusive, in part, because the pathogenesis ofDHF/DSS is not completely understood. While infection with one serotypeconfers lifelong homotypic immunity, it confers only short term(approximately three to six months) cross protection against heterotypicserotypes. Also, there is evidence that prior infection with one typecan produce an antibody response that can intensify, or enhance, thecourse of disease during a subsequent infection with a differentserotype. The possibility that vaccine components could elicit enhancingantibody responses, as opposed to protective responses, has been a majorconcern in designing and testing vaccines to protect against dengueinfections.

In late 2015 and early 2016, the first dengue vaccine, Dengvaxia(CYD-TDV) by Sanofi Pasteur, was registered in several countries for usein individuals 9-45 years of age living in endemic areas. Issues withthe vaccine include (1) weak protection against DENV1 and DENV2 (<60%efficacy); (2) relative risk of dengue hospitalization among children <9years old (7.5× higher than placebo); (3) immunogenicity not sustainedafter 1-2 years (implying the need for a 4^(th) dose booster); and (4)lowest efficacy against DENV2, which often causes more severeconditions. This latter point is a major weakness with the Dengvaxiavaccine, signaling the need of a new, more effective vaccine effectiveagainst DENV2. Other tetravalent live-attenuated vaccines are underdevelopment in phase II and phase III clinical trials, and other vaccinecandidates (based on subunit, DNA and purified inactivated virusplatforms) are at earlier stages of clinical development, although theability of these vaccine candidates to provide broad serotype protectionhas not been demonstrated.

Zika virus (ZIKV) is a member of the Flaviviridae virus family and theflavivirus genus. In humans, it causes a disease known as Zika fever. Itis related to dengue, yellow fever, West Nile and Japanese encephalitis,viruses that are also members of the virus family Flaviviridae. ZIKV isspread to people through mosquito bites. The most common symptoms ofZIKV disease (Zika) are fever, rash, joint pain, and red eye. Theillness is usually mild with symptoms lasting from several days to aweek. There is no vaccine to prevent, or medicine to treat, Zika virus.

Deoxyribonucleic acid (DNA) vaccination is one technique used tostimulate humoral and cellular immune responses to foreign antigens,such as ZIKV antigens. The direct injection of genetically engineeredDNA (e.g., naked plasmid DNA) into a living host results in a smallnumber of its cells directly producing an antigen, resulting in aprotective immunological response. With this technique, however, comespotential problems, including the possibility of insertionalmutagenesis, which could lead to the activation of oncogenes or theinhibition of tumor suppressor genes.

SUMMARY OF INVENTION

Provided herein is a ribonucleic acid (RNA) vaccine (e.g., messenger RNA(mRNA)) that can safely direct the body's cellular machinery to producenearly any protein of interest, from native proteins to antibodies andother entirely novel protein constructs that can have therapeuticactivity inside and outside of cells. The RNA vaccines of the presentdisclosure may be used to induce a balanced immune response against asingle virus or multiple viruses, including Chikungunya virus (CHIKV),Zika Virus (ZIKV) and Dengue virus (DENV), comprising both cellular andhumoral immunity, without the associated safety concerns, e.g., riskingthe possibility of insertional mutagenesis.

Some embodiments of the present disclosure provide vaccines and/orcombination vaccines comprising one or more RNA polynucleotides, e.g.,mRNA. In some embodiments, the RNA polynucleotide(s) encode a CHIKVantigen, a ZIKV antigen, a DENV antigen, or any combination of two orthree of the foregoing (e.g., CHIKV antigen/ZIKV antigen, CHIKVantigen/DENV antigen, ZIKV antigen/DENV antigen, or CHIKV/DENV/ZIKV) oneither the same polynucleotide or different polynucleotides. In someembodiments, the RNA polynucleotide(s) encode a ZIKV antigen and a DENVantigen, on either the same polynucleotide or different polynucleotides.

Thus, it should be understood the phrase “a CHIKV, DENV and/or ZIKV” isintended to encompass each individual virus in the alternative (CHIKV orDENV or ZIKV) as well as the individual combinations of CHIKV and DENV(CHIKV/DENV), CHIKV and ZIKV (CHIKV/ZIKV), ZIKV and DENV (ZIKV/DENV),and CHIKV, DENV and ZIKV (CHIKV/DENV/ZIKV).

In some aspects, the present disclosure provides a vaccine or acombination vaccine of at least one RNA polynucleotide encoding at leastone CHIKV antigenic polypeptide, at least one ZIKV antigenicpolypeptide, at least one DENV antigenic polypeptide, or a combinationof any two or three of the foregoing, and a pharmaceutically acceptablecarrier or excipient. In some embodiments, the RNA polynucleotidesencoding the DENV antigenic polypeptide, the ZIKV antigenic polypeptideand/or the CHIKV antigenic polypeptide are mono-cistronic RNApolynucleotides. In other embodiments, the RNA polynucleotide encodingthe DENV antigenic polypeptide, the ZIKV antigenic polypeptide and/orthe CHIKV antigenic polypeptide is a poly-cistronic. In otherembodiments, the RNA polynucleotides include combinations ofmono-cistronic and poly-cistronic RNA.

In some aspects, the present disclosure provides a vaccine or acombination vaccine of at least one RNA polynucleotide encoding at leastone ZIKV antigenic polypeptide and at least one DENV antigenicpolypeptide and a pharmaceutically acceptable carrier or excipient. Insome embodiments, the RNA polynucleotides encoding the ZIKV antigenicpolypeptide and the DENV antigenic polypeptide are mono-cistronic RNApolynucleotides. In other embodiments, the RNA polynucleotide encodingthe ZIKV antigenic polypeptide and the DENV antigenic polypeptide is apoly-cistronic RNA polynucleotide. In other embodiments, the RNApolynucleotides include combinations of mono-cistronic andpoly-cistronic RNA.

In some aspects, the present disclosure provides a vaccine or acombination vaccine of at least one RNA polynucleotide encoding at leastone ZIKV antigenic polypeptide and at least one CHIKV antigenicpolypeptide and a pharmaceutically acceptable carrier or excipient. Insome embodiments, the RNA polynucleotides encoding the ZIKV antigenicpolypeptide and the CHIKV antigenic polypeptide are mono-cistronic RNApolynucleotides. In other embodiments, the RNA polynucleotide encodingthe ZIKV antigenic polypeptide and the CHIKV antigenic polypeptide is apoly-cistronic RNA polynucleotide. In other embodiments, the RNApolynucleotides include combinations of mono-cistronic andpoly-cistronic RNA.

In some aspects, the present disclosure provides a vaccine or acombination vaccine of at least one RNA polynucleotide encoding at leastone DENV antigenic polypeptide and at least one CHIKV antigenicpolypeptide and a pharmaceutically acceptable carrier or excipient. Insome embodiments, the RNA polynucleotides encoding the DENV antigenicpolypeptide and the CHIKV antigenic polypeptide are mono-cistronic RNApolynucleotides. In other embodiments, the RNA polynucleotide encodingthe DENV antigenic polypeptide and the CHIKV antigenic polypeptide is apoly-cistronic RNA polynucleotide. In other embodiments, the RNApolynucleotides include combinations of mono-cistronic andpoly-cistronic RNA.

The at least one RNA polynucleotide, e.g., mRNA, in some embodiments,encodes two or more CHIKV antigenic polypeptides, two or more ZIKVantigenic polypeptides or two or more DENV antigenic polypeptides. Theat least one RNA polynucleotide, e.g., mRNA, in some embodiments,encodes two or more CHIKV antigenic polypeptides, two or more ZIKVantigenic polypeptides and two or more DENV antigenic polypeptides. Insome embodiments, the at least one RNA polynucleotide, e.g., mRNA,encodes two or more ZIKV antigenic polypeptides and two or more DENVantigenic polypeptides. In some embodiments, the at least one RNApolynucleotide, e.g., mRNA, encodes two or more ZIKV antigenicpolypeptides and two or more CHIKV antigenic polypeptides. In someembodiments, the at least one RNA polynucleotide, e.g., mRNA, encodestwo or more CHIKV antigenic polypeptides and two or more DENV antigenicpolypeptides.

The CHIKV antigenic polypeptide may be a Chikungunya structural proteinor an antigenic fragment or epitope thereof. The DENV antigenicpolypeptide may be a Dengue virus (DENV) structural protein or anantigenic fragment or epitope thereof. The ZIKV antigenic polypeptidemay be a Zika virus (ZIKV) structural protein (e.g., polyprotein) or anantigenic fragment or epitope thereof.

In some embodiments, the antigenic polypeptide is a CHIKV structuralprotein or an antigenic fragment thereof. For example, a CHIKVstructural protein may be an envelope protein (E), a 6K protein, or acapsid (C) protein. In some embodiments, the CHIKV structural protein isan envelope protein selected from E1, E2, and E3. In some embodiments,the CHIKV structural protein is E1 or E2. In some embodiments, the CHIKVstructural protein is a capsid protein. In some embodiments, theantigenic polypeptide is a fragment or epitope of a CHIKV structuralprotein.

In some embodiments, at least one antigenic polypeptide is a ZIKVpolyprotein. In some embodiments, at least one antigenic polypeptide isa ZIKV structural polyprotein. In some embodiments, at least oneantigenic polypeptide is a ZIKV nonstructural polyprotein.

In some embodiments, at least one antigenic polypeptide is a ZIKV capsidprotein, a ZIKV premembrane/membrane protein, a ZIKV envelope protein, aZIKV non-structural protein 1, a ZIKV non-structural protein 2A, a ZIKVnon-structural protein 2B, a ZIKV non-structural protein 3, a ZIKVnon-structural protein 4A, a ZIKV non-structural protein 4B, or a ZIKVnon-structural protein 5.

In some embodiments, at least one antigenic polypeptide is a ZIKV capsidprotein, a ZIKV premembrane/membrane protein, a ZIKV envelope protein, aZIKV non-structural protein 1, a ZIKV non-structural protein 2A, a ZIKVnon-structural protein 2B, a ZIKV non-structural protein 3, a ZIKVnon-structural protein 4A, a ZIKV non-structural protein 4B, or a ZIKVnon-structural protein 5.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV capsid protein, a RNApolynucleotide having an open reading frame encoding a ZIKVpremembrane/membrane protein, and a RNA polynucleotide having an openreading frame encoding a ZIKV envelope protein.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV capsid protein and a RNApolynucleotide having an open reading frame encoding a ZIKVpremembrane/membrane protein.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV capsid protein and a RNApolynucleotide having an open reading frame encoding a ZIKV envelopeprotein.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV premembrane/membrane protein and aRNA polynucleotide having an open reading frame encoding a ZIKV envelopeprotein.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV capsid protein and at least oneRNA polynucleotide having an open reading frame encoding any one or moreof a ZIKV non-structural protein 1, 2A, 2B, 3, 4A, 4B or 5.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV premembrane/membrane protein andat least one RNA polynucleotide having an open reading frame encodingany one or more of a ZIKV non-structural protein 1, 2A, 2B, 3, 4A, 4B or5.

In some embodiments, the vaccine comprises a RNA polynucleotide havingan open reading frame encoding a ZIKV envelope protein and at least oneRNA polynucleotide having an open reading frame encoding any one or moreof a ZIKV non-structural protein 1, 2A, 2B, 3, 4A, 4B or 5.

In some embodiments, the at least one antigenic polypeptide comprises acombination of any two or more of a ZIKV capsid protein, a ZIKVpremembrane/membrane protein, a ZIKV envelope protein, a ZIKVnon-structural protein 1, a ZIKV non-structural protein 2A, a ZIKVnon-structural protein 2B, a ZIKV non-structural protein 3, a ZIKVnon-structural protein 4A, a ZIKV non-structural protein 4B, or a ZIKVnon-structural protein 5.

In some embodiments, the at least one ZIKV antigenic polypeptide isfused to signal peptide having a sequence set forth as SEQ ID NO: 125,126, 128 or 131. In some embodiments, the signal peptide is fused to theN-terminus of the at least one ZIKV antigenic polypeptide.

In some embodiments, the antigenic polypeptide comprises two or moreCHIKV structural proteins. In some embodiments, the two or more CHIKVstructural proteins are envelope proteins. In some embodiments, the twoor more CHIKV structural proteins are E1 and E2. In some embodiments,the two or more CHIKV structural proteins are E1 and E3. In someembodiments, the two or more CHIKV structural proteins are E2 and E3. Insome embodiments, the two or more CHIKV structural proteins are E1, E2,and E3. In some embodiments, the two or more CHIKV structural proteinsare envelope and capsid proteins. In some embodiments, the two or moreCHIKV structural proteins are E1 and C. In some embodiments, the two ormore CHIKV structural proteins are E2 and C. In some embodiments, thetwo or more CHIKV structural proteins are E3 and C. In some embodiments,the two or more CHIKV structural proteins are E1, E2, and C. In someembodiments, the two or more CHIKV structural proteins are E1, E3, andC. In some embodiments, the two or more CHIKV structural proteins areE2, E3, and C. In some embodiments, the two or more CHIKV structuralproteins are E1, E2, E3, and C. In some embodiments, the two or moreCHIKV structural proteins are E1, 6K, and E2. In some embodiments, thetwo or more CHIKV structural proteins are E2, 6K, and E3. In someembodiments, the two or more CHIKV structural proteins are E1, 6K, andE3. In some embodiments, the two or more CHIKV structural proteins areE1, E2, E3, 6K, and C. In some embodiments, the antigenic polypeptidecomprises the CHIKV structural polyprotein comprising C, E3, E2, 6K, andE1. In some embodiments, the antigenic polypeptide is a fragment orepitope of two or more CHIKV structural proteins or a fragment orepitope of the polyprotein.

In some embodiments the at least one antigenic polypeptide has greaterthan 90% identity to an amino acid sequence of any one of Tables 13, 15,18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15,17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and hasmembrane fusion activity. In some embodiments the at least one CHIKVantigenic polypeptide has greater than 90% identity to an amino acidsequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusionactivity. In some embodiments the at least one DENV antigenicpolypeptide has greater than 90% identity to an amino acid sequence ofany one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, 162-298 and hasmembrane fusion activity. In some embodiments the at least one ZIKVantigenic polypeptide has greater than 90% identity to an amino acidsequence of any one of SEQ ID NO: 67-134 and has membrane fusionactivity.

In some embodiments the at least one antigenic polypeptide has greaterthan 95% identity to an amino acid sequence of any one of Tables 13, 15,18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15,17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and hasmembrane fusion activity. In some embodiments the at least one CHIKVantigenic polypeptide has greater than 95% identity to an amino acidsequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusionactivity. In some embodiments the at least one DENV antigenicpolypeptide has greater than 95% identity to an amino acid sequence ofany one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, or 162-298 and hasmembrane fusion activity. In some embodiments the at least one ZIKVantigenic polypeptide has greater than 95% identity to an amino acidsequence of any one of SEQ ID NO: 67-134 and has membrane fusionactivity.

In some embodiments the at least one antigenic polypeptide has greaterthan 96% identity to an amino acid sequence of any one of Tables 13, 15,18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15,17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and hasmembrane fusion activity. In some embodiments the at least one CHIKVantigenic polypeptide has greater than 96% identity to an amino acidsequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusionactivity. In some embodiments the at least one DENV antigenicpolypeptide has greater than 96% identity to an amino acid sequence ofany one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, or 162-298 and hasmembrane fusion activity. In some embodiments the at least one ZIKVantigenic polypeptide has greater than 96% identity to an amino acidsequence of any one of SEQ ID NO: 67-134 and has membrane fusionactivity.

In some embodiments the at least one antigenic polypeptide has greaterthan 97% identity to an amino acid sequence of any one of Tables 13, 15,18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15,17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and hasmembrane fusion activity. In some embodiments the at least one CHIKVantigenic polypeptide has greater than 97% identity to an amino acidsequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusionactivity. In some embodiments the at least one DENV antigenicpolypeptide has greater than 97% identity to an amino acid sequence ofany one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, or 162-298 and hasmembrane fusion activity. In some embodiments the at least one ZIKVantigenic polypeptide has greater than 97% identity to an amino acidsequence of any one of SEQ ID NO: 67-134 and has membrane fusionactivity.

In some embodiments the at least one antigenic polypeptide has greaterthan 98% identity to an amino acid sequence of any one of Tables 13, 15,18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15,17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and hasmembrane fusion activity. In some embodiments the at least one CHIKVantigenic polypeptide has greater than 98% identity to an amino acidsequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusionactivity. In some embodiments the at least one DENV antigenicpolypeptide has greater than 98% identity to an amino acid sequence ofany one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, or 162-298 and hasmembrane fusion activity. In some embodiments the at least one ZIKVantigenic polypeptide has greater than 98% identity to an amino acidsequence of any one of SEQ ID NO: 67-134 and has membrane fusionactivity.

In some embodiments the at least one antigenic polypeptide has greaterthan 99% identity to an amino acid sequence of any one of Tables 13, 15,18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15,17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and hasmembrane fusion activity. In some embodiments the at least one CHIKVantigenic polypeptide has greater than 99% identity to an amino acidsequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusionactivity. In some embodiments the at least one DENV antigenicpolypeptide has greater than 99% identity to an amino acid sequence ofany one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, or 162-298 and hasmembrane fusion activity. In some embodiments the at least one ZIKVantigenic polypeptide has greater than 99% identity to an amino acidsequence of any one of SEQ ID NO: 67-134 and has membrane fusionactivity.

In some embodiments the at least one antigenic polypeptide has greaterthan 95-99% identity to an amino acid sequence of any one of Tables 13,15, 18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV),15, 17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and hasmembrane fusion activity. In some embodiments the at least one CHIKVantigenic polypeptide has greater than 95-99% identity to an amino acidsequence of any one of SEQ ID NO: 14 or 37-47 and has membrane fusionactivity. In some embodiments the at least one DENV antigenicpolypeptide has greater than 95-99% identity to an amino acid sequenceof any one of SEQ ID NO: 15, 17, 19, 21, 23, 26, 29, 32, or 162-298 andhas membrane fusion activity. In some embodiments the at least one ZIKVantigenic polypeptide has greater than 95-99% identity to an amino acidsequence of any one of SEQ ID NO: 67-134 and has membrane fusionactivity.

In other embodiments the at least one antigenic polypeptides encode anantigenic polypeptide having an amino acid sequence of Tables 13, 15,18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15,17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and whereinthe RNA polynucleotide is codon optimized mRNA. In yet other embodimentsthe at least one antigenic polypeptide has an amino acid sequence ofTables 13, 15, 18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47(CHIKV), 15, 17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134(ZIKV) and wherein the RNA polynucleotide has less than 80% identity towild-type mRNA sequence. According to some embodiments the at least oneantigenic polypeptide has an amino acid sequence of Tables 13, 15,18-27, 32 or 34-37, or any one of SEQ ID NO: 14 or 37-47 (CHIKV), 15,17, 19, 21, 23, 26, 29, 32, 162-298 (DENV), or 67-134 (ZIKV) and whereinthe RNA polynucleotide has greater than 80% identity to wild-type mRNAsequence, but does not include wild-type mRNA sequence.

In some embodiments, the DENV antigen is a concatemeric DENV antigen. Insome embodiments, the DENV concatemeric antigen comprises between 2-100DENV peptide epitopes connected directly to one another or interspersedby linkers. In some embodiments, the DENV vaccine's peptide epitopes areT cell epitopes and/or B cell epitopes. In other embodiments, the DENVvaccine's peptide epitopes comprise a combination of T cell epitopes andB cell epitopes. In some embodiments, at least one of the peptideepitopes of the DENV vaccine is a T cell epitope. In some embodiments,at least one of the peptide epitopes of the DENV vaccine is a B cellepitope. In some embodiments, the T cell epitope of the DENV vaccinecomprises between 8-11 amino acids. In some embodiments, the B cellepitope of the DENV vaccine comprises between 13-17 amino acids.

In some embodiments, the RNA polynucleotide, e.g., mRNA, of a vaccine isencoded by at least one polynucleotide comprising a nucleotide sequencehaving at least 80%, 85%, 90%, 95%, 98% or 99% identity to any of thenucleotide sequences of Tables 1-4, 13, 15, 31, 34 or 38, or any one ofSEQ ID NO: 1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34,144-152 or 199-212 (DENV), or 48-66 (ZIKV). In some embodiments, the RNApolynucleotide, e.g., mRNA, of a vaccine is encoded by at least onepolynucleotide comprising a nucleotide sequence having at least 80%,85%, 90%, 95%, 98% or 99% identity to any of the CHIKV nucleotidesequences of SEQ ID NO: 1-13. In some embodiments, the RNApolynucleotide, e.g., mRNA, of a vaccine is encoded by at least onepolynucleotide comprising a nucleotide sequence having at least 80%,85%, 90%, 95%, 98% or 99% identity to any of the DENV nucleotidesequences of SEQ ID NO: 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34,144-152 or 199-212. In some embodiments, the RNA polynucleotide, e.g.,mRNA, of a vaccine is encoded by at least one polynucleotide comprisinga nucleotide sequence having at least 80%, 85%, 90%, 95%, 98% or 99%identity to any of the ZIKV nucleotide sequences of SEQ ID NO: 67-134.

In other embodiments, the RNA polynucleotide comprises a polynucleotidesequence derived from an Asian strain, Brazilian strain, West Africanstrain, ECSA strain, and Indian Ocean strain of Chikungunya.

In some embodiments, at least one antigenic polypeptide is a ZIKVenvelope protein.

In some embodiments, at least one antigenic polypeptide is a Spondwenivirus Polyprotein.

In some embodiments, at least one antigenic polypeptide is a polyproteinobtained from ZIKV strain MR 766, ACD75819 or SPH2015.

In some embodiments, at least one antigenic polypeptide has an aminoacid sequence of any one of the sequences listed in Table 32.

In some embodiments, at least one antigenic polypeptide has at least 95%identity to an antigenic polypeptide having an amino acid sequence ofany one of the sequences listed in Table 32.

In some embodiments, the at least one RNA polynucleotide encodes atleast one antigenic polypeptide having a sequence of listed in Table 31.

In some embodiments, the at least one RNA polynucleotide encodes atleast one protein variant having at least 95% identity to an antigenicpolypeptide having a sequence of listed in Table 31.

Tables herein provide National Center for Biotechnology Information(NCBI) accession numbers of interest. It should be understood that thephrase “an amino acid sequence of Table X” (e.g., Table 33 or Table 35)refers to an amino acid sequence identified by one or more NCBIaccession numbers listed in Table X. Each of the amino acid sequences,and variants having greater than 95% identity to each of the amino acidsequences encompassed by the accession numbers of Table X (e.g., Table33 or Table 35) are included within the constructs of the presentdisclosure.

In some embodiments, at least one RNA polynucleotide encodes anantigenic polypeptide having at least 90% identity to an amino acidsequence of Table 32 or 33 Table 32 or 33 and having membrane fusionactivity. In some embodiments, at least one RNA polynucleotide encodesan antigenic polypeptide having at least 95% identity to an amino acidsequence of Table 32 or 33 and having membrane fusion activity. In someembodiments, at least one RNA polynucleotide encodes an antigenicpolypeptide having at least 96% identity to an amino acid sequence ofTable 32 or 33 and having membrane fusion activity. In some embodiments,at least one RNA polynucleotide encodes an antigenic polypeptide havingat least 97% identity to an amino acid sequence of Table 32 or 33 andhaving membrane fusion activity. In some embodiments, at least one RNApolynucleotide encodes an antigenic polypeptide having at least 98%identity to an amino acid sequence of Table 32 or 33 and having membranefusion activity. In some embodiments, at least one RNA polynucleotideencodes an antigenic polypeptide having at least 99% identity to anamino acid sequence of Table 32 or 33 and having membrane fusionactivity. In some embodiments, at least one RNA polynucleotide encodesan antigenic polypeptide having 95-99% identity to an amino acidsequence of Table 32 or 33 and having membrane fusion activity.

In some embodiments, at least one RNA polynucleotide encodes anantigenic polypeptide having an amino acid sequence of Table 32 or 33and is codon optimized mRNA.

In some embodiments, at least one RNA polynucleotide encodes anantigenic polypeptide having an amino acid sequence of Table 32 or 33and has less than 80% identity to wild-type mRNA sequence. In someembodiments, at least one RNA polynucleotide encodes an antigenicpolypeptide having an amino acid sequence of Table 32 or 33 and has lessthan 75%, 85% or 95% identity to wild-type mRNA sequence. In someembodiments, at least one RNA polynucleotide encodes an antigenicpolypeptide having an amino acid sequence of Table 32 or 33 and has50-80%, 60-80%, 40-80%, 30-80%, 70-80%, 75-80% or 78-80% identity towild-type mRNA sequence. In some embodiments, at least one RNApolynucleotide encodes an antigenic polypeptide having an amino acidsequence of Table 32 or 33 and has 40-85%, 50-85%, 60-85%, 30-85%,70-85%, 75-85%, or 80-85% identity to wild-type mRNA sequence. In someembodiments, at least one RNA polynucleotide encodes an antigenicpolypeptide having an amino acid sequence of Table 32 or 33 and has40-90%, 50-90%, 60-90%, 30-90%, 70-90%, 75-90%, 80-90%, or 85-90%identity to wild-type mRNA sequence.

In some embodiments, at least one RNA polynucleotide is encoded by anucleic acid having at least 90% identity to a nucleic acid sequence ofTable 31. In some embodiments, at least one RNA polynucleotide isencoded by a nucleic acid having at least 95% identity to a nucleic acidsequence of Table 31. In some embodiments, at least one RNApolynucleotide is encoded by a nucleic acid having at least 96% identityto a nucleic acid sequence of Table 31. In some embodiments, at leastone RNA polynucleotide is encoded by a nucleic acid having at least 97%identity to a nucleic acid sequence of Table 31. In some embodiments, atleast one RNA polynucleotide is encoded by a nucleic acid having atleast 98% identity to a nucleic acid sequence of Table 31. In someembodiments, at least one RNA polynucleotide is encoded by a nucleicacid having at least 99% identity to a nucleic acid sequence of Table31. In some embodiments, at least one RNA polynucleotide is encoded by anucleic acid having 95-99% identity to a nucleic acid sequence of Table31.

In some embodiments, at least one mRNA polynucleotide is encoded by anucleic acid having a sequence of Table 31 and has less than 80%identity to wild-type mRNA sequence. In some embodiments, at least onemRNA polynucleotide is encoded by a nucleic acid having a sequence ofTable 31 and has less than 75%, 85% or 95% identity to a wild-type mRNAsequence. In some embodiments, at least one mRNA polynucleotide isencoded by a nucleic acid having a sequence of Table 31 and has lessthan 50-80%, 60-80%, 40-80%, 30-80%, 70-80%, 75-80% or 78-80% identityto wild-type mRNA sequence. In some embodiments, at least one mRNApolynucleotide is encoded by a nucleic acid having a sequence of Table31 and has less than 40-85%, 50-85%, 60-85%, 30-85%, 70-85%, 75-85% or80-85% identity to wild-type mRNA sequence. In some embodiments, atleast one mRNA polynucleotide is encoded by a nucleic acid having asequence of Table 31 and has less than 40-90%, 50-90%, 60-90%, 30-90%,70-90%, 75-90%, 80-90%, or 85-90% identity to wild-type mRNA sequence.

In some embodiments, at least one RNA polynucleotide encodes anantigenic polypeptide having an amino acid sequence of Table 32 or 33and having at least 80% identity to wild-type mRNA sequence, but doesnot include wild-type mRNA sequence.

In some embodiments, at least one RNA polynucleotide encodes anantigenic polypeptide that attaches to cell receptors.

In some embodiments, at least one RNA polynucleotide encodes anantigenic polypeptide that causes fusion of viral and cellularmembranes.

In some embodiments, at least one RNA polynucleotide encodes anantigenic polypeptide that is responsible for binding of the ZIKV to acell being infected.

Some embodiments of the present disclosure provide a CHIKV vaccine thatincludes at least one RNA polynucleotide having an open reading frameencoding a CHIKV antigenic polypeptides, in which the RNA polynucleotideof the CHIKV vaccine includes a 5′ terminal cap. Some embodiments of thepresent disclosure provide a DENV vaccine that includes at least one RNApolynucleotide having an open reading frame encoding a DENV antigenicpolypeptides, in which the RNA polynucleotide of the DENV vaccineincludes a 5′ terminal cap. Some embodiments of the present disclosureprovide a ZIKV vaccine that includes at least one RNA polynucleotidehaving an open reading frame encoding a ZIKV antigenic polypeptides, inwhich the RNA polynucleotide of the ZIKV vaccine includes a 5′ terminalcap.

Some embodiments of the present disclosure provide a CHIKV/DENV/ZIKVcombination vaccine that includes at least one RNA polynucleotide havingan open reading frame encoding at least one each of CHIKV, DENV, andZIKV antigenic polypeptides, in which the RNA polynucleotide of theCHIKV, DENV, and ZIKV RNA vaccine includes a 5′ terminal cap. Someembodiments of the present disclosure provide a DENV/ZIKV combinationvaccine that includes at least one RNA polynucleotide having an openreading frame encoding at least one each of DENV and ZIKV antigenicpolypeptides, in which the RNA polynucleotide of the DENV, and ZIKV RNAvaccine includes a 5′ terminal cap. Some embodiments of the presentdisclosure provide a CHIKV/ZIKV combination vaccine that includes atleast one RNA polynucleotide having an open reading frame encoding atleast one each of CHIKV and ZIKV antigenic polypeptides, in which theRNA polynucleotide of the CHIKV and ZIKV RNA vaccine includes a 5′terminal cap. Some embodiments of the present disclosure provide aCHIKV/DENV combination vaccine that includes at least one RNApolynucleotide having an open reading frame encoding at least one eachof CHIKV and DENV antigenic polypeptides, in which the RNApolynucleotide of the CHIKV and DENV RNA vaccine includes a 5′ terminalcap. In some embodiments, the 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp.

Some embodiments of the present disclosure provide a vaccine thatincludes at least one RNA polynucleotide having an open reading frameencoding at least one CHIKV antigenic polypeptide in which the RNApolynucleotide of the CHIKV RNA vaccine includes at least one chemicalmodification. Some embodiments of the present disclosure provide avaccine that includes at least one RNA polynucleotide having an openreading frame encoding at least one DENV antigenic polypeptide in whichthe RNA polynucleotide of the DENV RNA vaccine includes at least onechemical modification. Some embodiments of the present disclosureprovide a vaccine that includes at least one RNA polynucleotide havingan open reading frame encoding at least one ZIKV antigenic polypeptidein which the RNA polynucleotide of the ZIKV RNA vaccine includes atleast one chemical modification.

Some embodiments of the present disclosure provide a combination vaccinethat includes at least one RNA polynucleotide having an open readingframe encoding at least one CHIKV antigenic polypeptide, at least oneDENV antigenic polypeptide, and at least one ZIKV antigenic polypeptidein which the RNA polynucleotide of the CHIKV/DENV/ZIKV combination RNAvaccine includes at least one chemical modification. Some embodiments ofthe present disclosure provide a combination vaccine that includes atleast one RNA polynucleotide having an open reading frame encoding atleast one CHIKV antigenic polypeptide and at least one DENV antigenicpolypeptide in which the RNA polynucleotide of the CHIKV/DENVcombination RNA vaccine includes at least one chemical modification.Some embodiments of the present disclosure provide a combination vaccinethat includes at least one RNA polynucleotide having an open readingframe encoding at least one CHIKV antigenic polypeptide and at least oneZIKV antigenic polypeptide in which the RNA polynucleotide of theCHIKV/ZIKV combination RNA vaccine includes at least one chemicalmodification. Some embodiments of the present disclosure provide acombination vaccine that includes at least one RNA polynucleotide havingan open reading frame encoding at least one DENV antigenic polypeptideand at least one ZIKV antigenic polypeptide in which the RNApolynucleotide of the DENV/ZIKV combination RNA vaccine includes atleast one chemical modification.

In some embodiments, the chemical modification is selected frompseudouridine, N1-methylpseudouridine, 2-thiouridine, 4′-thiouridine,5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methoxyuridine, 5-methyluridine, and 2′-O-methyl uridine.

In some embodiments, the RNA polynucleotide, e.g., mRNA including atleast one chemical modification further includes a 5′ terminal cap. Insome embodiments, the 5′ terminal cap is 7mG(5′)ppp(5′)NlmpNp.

Some embodiments of the present disclosure provide a vaccine thatincludes at least one RNA polynucleotide having an open reading frameencoding at least one CHIKV antigenic polypeptide, wherein at least 80%of the uracil in the open reading frame have a chemical modification.Some embodiments of the present disclosure provide a vaccine thatincludes at least one RNA polynucleotide having an open reading frameencoding at least one DENV antigenic polypeptide, wherein at least 80%of the uracil in the open reading frame have a chemical modification.Some embodiments of the present disclosure provide a vaccine thatincludes at least one RNA polynucleotide having an open reading frameencoding at least one ZIKV antigenic polypeptide, wherein at least 80%of the uracil in the open reading frame have a chemical modification.

Some embodiments of the present disclosure provide a combination vaccinethat includes at least one RNA polynucleotide having an open readingframe encoding at least one CHIKV antigenic polypeptide and at least oneDENV antigenic polypeptide, wherein at least 80% of the uracil in theopen reading frame have a chemical modification. Some embodiments of thepresent disclosure provide a combination vaccine that includes at leastone RNA polynucleotide having an open reading frame encoding at leastone CHIKV antigenic polypeptide and at least one ZIKV antigenicpolypeptide, wherein at least 80% of the uracil in the open readingframe have a chemical modification. Some embodiments of the presentdisclosure provide a combination vaccine that includes at least one RNApolynucleotide having an open reading frame encoding at least one DENVantigenic polypeptide and at least one ZIKV antigenic polypeptide,wherein at least 80% of the uracil in the open reading frame have achemical modification. Some embodiments of the present disclosureprovide a combination vaccine that includes at least one RNApolynucleotide having an open reading frame encoding at least one CHIKVantigenic polypeptide, at least one DENV antigenic polypeptide, and atleast one ZIKV antigenic polypeptide, wherein at least 80% of the uracilin the open reading frame have a chemical modification. In someembodiments, 100% of the uracil in the open reading frame have achemical modification. In some embodiments, the chemical modification isin the 5-position of the uracil. In some embodiments, the chemicalmodification is a N1-methyl pseudouridine.

In some embodiments of any of the combination RNA vaccines describedherein, the RNA polynucleotide of the RNA vaccine is formulated in alipid nanoparticle (LNP) carrier. In some embodiments, the lipidnanoparticle comprises a cationic lipid, a PEG-modified lipid, a steroland a non-cationic lipid. In some embodiments, the lipid nanoparticlecarrier comprising a molar ratio of about 20-60% cationic lipid: 5-25%non-cationic lipid: 25-55% sterol; and 0.5-15% PEG-modified lipid. Insome embodiments, the cationic lipid is an ionizable cationic lipid. Insome embodiments, the non-cationic lipid is a neutral lipid. In someembodiments, the sterol is a cholesterol. In some embodiments, thecationic lipid is selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). In someembodiments, the lipid nanoparticle has a polydispersity value of lessthan 0.4. In some embodiments, the lipid nanoparticle has a net neutralcharge at a neutral pH. In some embodiments, the lipid nanoparticle hasa mean diameter of 50-200 nm.

Some embodiments of the present disclosure provide a vaccine thatincludes at least one RNA polynucleotide having an open reading frameencoding at least one CHIKV antigenic polypeptide, at least one 5′terminal cap and at least one chemical modification, formulated within alipid nanoparticle. Some embodiments of the present disclosure provide avaccine that includes at least one RNA polynucleotide having an openreading frame encoding at least one DENV antigenic polypeptide, at leastone 5′ terminal cap and at least one chemical modification, formulatedwithin a lipid nanoparticle. Some embodiments of the present disclosureprovide a vaccine that includes at least one RNA polynucleotide havingan open reading frame encoding at least one ZIKV antigenic polypeptide,at least one 5′ terminal cap and at least one chemical modification,formulated within a lipid nanoparticle.

Some embodiments of the present disclosure provide a combination vaccinethat includes at least one RNA polynucleotide having an open readingframe encoding at least one CHIKV antigenic polypeptide and at least oneDENV antigenic polypeptide, at least one 5′ terminal cap and at leastone chemical modification, formulated within a lipid nanoparticle. Someembodiments of the present disclosure provide a combination vaccine thatincludes at least one RNA polynucleotide having an open reading frameencoding at least one CHIKV antigenic polypeptide and at least one ZIKVantigenic polypeptide, at least one 5′ terminal cap and at least onechemical modification, formulated within a lipid nanoparticle. Someembodiments of the present disclosure provide a combination vaccine thatincludes at least one RNA polynucleotide having an open reading frameencoding at least one ZIKV antigenic polypeptide and at least one DENVantigenic polypeptide, at least one 5′ terminal cap and at least onechemical modification, formulated within a lipid nanoparticle. Someembodiments of the present disclosure provide a combination vaccine thatincludes at least one RNA polynucleotide having an open reading frameencoding at least one CHIKV antigenic polypeptide, at least one DENVantigenic polypeptide, at least one ZIKV antigenic polypeptide, at leastone 5′ terminal cap and at least one chemical modification, formulatedwithin a lipid nanoparticle.

Some embodiments of the present disclosure provide a vaccine thatincludes at least one RNA polynucleotide having an open reading frameencoding at least one CHIKV antigenic polypeptide, wherein the openreading frame of the RNA polynucleotide is codon-optimized. Someembodiments of the present disclosure provide a vaccine that includes atleast one RNA polynucleotide having an open reading frame encoding atleast one DENV antigenic polypeptide, wherein the open reading frame ofthe RNA polynucleotide is codon-optimized. Some embodiments of thepresent disclosure provide a vaccine that includes at least one RNApolynucleotide having an open reading frame encoding at least one ZIKVantigenic polypeptide, wherein the open reading frame of the RNApolynucleotide is codon-optimized.

Some embodiments of the present disclosure provide a combination vaccinethat includes at least one RNA polynucleotide having an open readingframe encoding at least one CHIKV antigenic polypeptide and at least oneDENV antigenic polypeptide, wherein the open reading frame of the RNApolynucleotide is codon-optimized. Some embodiments of the presentdisclosure provide a combination vaccine that includes at least one RNApolynucleotide having an open reading frame encoding at least one CHIKVantigenic polypeptide and at least one ZIKV antigenic polypeptide,wherein the open reading frame of the RNA polynucleotide iscodon-optimized. Some embodiments of the present disclosure provide acombination vaccine that includes at least one RNA polynucleotide havingan open reading frame encoding at least one ZIKV antigenic polypeptideand at least one DENV antigenic polypeptide, wherein the open readingframe of the RNA polynucleotide is codon-optimized. Some embodiments ofthe present disclosure provide a combination vaccine that includes atleast one RNA polynucleotide having an open reading frame encoding atleast one CHIKV antigenic polypeptide, at least one DENV antigenicpolypeptide, and at least one ZIKV antigenic polypeptide, wherein theopen reading frame of the RNA polynucleotide is codon-optimized.

Some embodiments of the present disclosure provide methods of inducingan antigen specific immune response in a subject, comprisingadministering to the subject a combination RNA vaccine in an amounteffective to produce an antigen specific immune response against CHIKV,against DENV, against ZIKV, against CHIKV and DENV, against CHIKV andZIKV, against DENV and ZIKV, or against CHIKV, DENV and ZIKV. In someembodiments, an antigen specific immune response comprises a T cellresponse. In some embodiments, an antigen specific immune responsecomprises a B cell response. In some embodiments, an antigen specificimmune response comprises both a T cell response and a B cell response.In some embodiments, a method of producing an antigen specific immuneresponse involves a single administration of the vaccine. In otherembodiments, the method further comprises administering to the subject asecond dose or a booster dose of the vaccine. In other embodiments themethod comprises administering more than one dose of the vaccine, forexample, 2, 3, 4 or more doses of the vaccine. In some embodiments, thevaccine is administered to the subject by intradermal or intramuscularinjection.

Further provided herein are vaccines, such as any of the vaccinesdescribed herein, for use in a method of inducing an antigen specificimmune response in a subject, the method comprising administering thevaccine to the subject in an effective amount to produce an antigenspecific immune response.

Further provided herein are uses of CHIKV, DENV or ZIKV RNA vaccines andCHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV or CHIKV/DENV/ZIKV combination RNAvaccines in the manufacture of a medicament for use in a method ofinducing an antigen specific immune response in a subject, the methodcomprising administering the vaccine to the subject in an amounteffective to produce an antigen specific immune response.

In other aspects of the invention is a method of preventing or treatinga CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, orCHIKV/DENV/ZIKV infection comprising administering to a subject any ofthe vaccines described herein. In yet other aspects of the invention isa method of preventing or treating CHIKV, DENV, ZIKV, CHIKV/DENV,CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV

In some embodiments, a CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV,DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine, is formulated in an effectiveamount to produce an antigen specific immune response in a subject.

In some embodiments, an anti-CHIKV, an anti-DENV, an anti-ZIKV, ananti-CHIKV/anti-DENV, an anti-CHIKV/anti-ZIKV, an anti-DENV/anti-ZIKV,or an anti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibodytiter produced in the subject is increased by at least 1 log relative toa control. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or ananti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is increased by 1-3 log relative to a control.In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or ananti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is increased at least 2 times relative to acontrol. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or ananti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is increased at least 5 times relative to acontrol. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or ananti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is increased at least 10 times relative to acontrol. In some embodiments, the anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or ananti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is increased 2-10 times relative to a control.

In some embodiments, the control is an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, or ananti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in a subject who has not been administered a combination (orany other) vaccine. In some embodiments, the control is an anti-CHIKV,anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or an anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a subject who has beenadministered a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV, vaccine. In someembodiments, the control is an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in a subject who has been administered a recombinant orpurified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, orCHIKV/DENV/ZIKV protein vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 2-fold reduction in the standard of care dose of a recombinantCHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKVprotein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is equivalent to an anti-CHIKV, anti-DENV,anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified CHIKV, DENV,ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV proteinvaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 4-fold reduction in the standard of care dose of a recombinantCHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKVprotein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is equivalent to an anti-CHIKV, anti-DENV,anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified CHIKV, DENV,ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV proteinvaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 10-fold reduction in the standard of care dose of a recombinantCHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKVprotein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is equivalent to an anti-CHIKV, anti-DENV,anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified CHIKV, DENV,ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV proteinvaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 100-fold reduction in the standard of care dose of a recombinantCHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKVprotein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is equivalent to an anti-CHIKV, anti-DENV,anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified CHIKV, DENV,ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV proteinvaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 1000-fold reduction in the standard of care dose of a recombinantCHIKV/DENV/ZIKV, or DENV/ZIKV, protein vaccine, and wherein ananti-CHIKV, anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV,anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is equivalent to an anti-CHIKV, anti-DENV,anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified CHIKV, DENV,ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV proteinvaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.

In some embodiments, the effective amount is a dose equivalent to a2-1000-fold reduction in the standard of care dose of a recombinantCHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKVprotein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is equivalent to an anti-CHIKV, anti-DENV,anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified CHIKV, DENV,ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV proteinvaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.

In some embodiments, the effective amount is a total dose of 50-1000 μg.In some embodiments, the effective amount is a total dose of 100 μg. Insome embodiments, the effective amount is a dose of 25 μg administeredto the subject a total of two times. In some embodiments, the effectiveamount is a dose of 100 μg administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 400 μgadministered to the subject a total of two times. In some embodiments,the effective amount is a dose of 500 μg administered to the subject atotal of two times.

Further provided herein is a method of inducing an antigen specificimmune response in a subject, the method including administering to asubject the CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, orCHIKV/DENV/ZIKV vaccine in an effective amount to produce an antigenspecific immune response in a subject. In some embodiments, anti-CHIKV,anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV, antigenicpolypeptide antibody titer produced in the subject is increased by atleast 1 log relative to a control. In some embodiments, an anti-CHIKV,anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV, antigenicpolypeptide antibody titer produced in the subject is increased by 1-3log relative to a control. In some embodiments, the anti-CHIKV,anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV, antigenicpolypeptide antibody titer produced in the subject is increased at least2 times relative to a control. In some embodiments, the anti-CHIKV,anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV, antigenicpolypeptide antibody titer produced in the subject is increased at least5 times relative to a control. In some embodiments, the anti-CHIKV,anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in the subject is increased at least10 times relative to a control. In some embodiments, the anti-CHIKV,anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in the subject is increased 2-10times relative to a control.

In some embodiments, the control is an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in a subject who has not been administered CHIKV/DENV/ZIKV, orDENV/ZIKV, vaccine. In some embodiments, the control is an anti-CHIKV,anti-DENV, anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a subject who has beenadministered a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine. In someembodiments, the control is an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in a subject who has been administered a recombinant orpurified CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, orCHIKV/DENV/ZIKV protein vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 2-fold reduction in the standard of care dose of a recombinantCHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKVprotein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is equivalent to an anti-CHIKV, anti-DENV,anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein vaccine ora live attenuated CHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV,or CHIKV/DENV/ZIKV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 4-fold reduction in the standard of care dose of a recombinantCHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKVprotein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is equivalent to anti-CHIKV, anti-DENV,anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified CHIKV, DENV,ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV proteinvaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 10-fold reduction in the standard of care dose of a recombinantCHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKVprotein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is equivalent to an anti-CHIKV, anti-DENV,anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHI

KV/anti-ZIKV, anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purifiedCHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKVprotein vaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 100-fold reduction in the standard of care dose of a recombinantCHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKVprotein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is equivalent to an anti-CHIKV, anti-DENV,anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified CHIKV, DENV,ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV proteinvaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.

In some embodiments, the effective amount is a dose equivalent to an atleast 1000-fold reduction in the standard of care dose of a recombinantCHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKVprotein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is equivalent to an anti-CHIKV, anti-DENV,anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified CHIKV, DENV,ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV proteinvaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.

In some embodiments, wherein the effective amount is a dose equivalentto a 2-1000-fold reduction in the standard of care dose of a recombinantCHIKV, DENV, ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKVprotein vaccine, and wherein an anti-CHIKV, anti-DENV, anti-ZIKV,anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV, anti-DENV/anti-ZIKV, oranti-CHIKV/anti-DENV/anti-ZIKV antigenic polypeptide antibody titerproduced in the subject is equivalent to an anti-CHIKV, anti-DENV,anti-ZIKV, anti-CHIKV/anti-DENV, anti-CHIKV/anti-ZIKV,anti-DENV/anti-ZIKV, or anti-CHIKV/anti-DENV/anti-ZIKV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified CHIKV, DENV,ZIKV, CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV, proteinvaccine or a live attenuated or inactivated CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.

In some embodiments, the effective amount is a total dose of 50-1000 μg.In some embodiments, the effective amount is a total dose of 100 μg. Insome embodiments, the effective amount is a dose of 25 μg administeredto the subject a total of two times. In some embodiments, the effectiveamount is a dose of 100 μg administered to the subject a total of twotimes. In some embodiments, the effective amount is a dose of 400 μgadministered to the subject a total of two times. In some embodiments,the effective amount is a dose of 500 μg administered to the subject atotal of two times.

Other aspects of the present disclosure provide a CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine, whichincludes a signal peptide linked to a CHIKV, DENV, ZIKV, CHIKV/DENV,CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV protein. In some embodiments,the signal peptide is a IgE signal peptide. In some embodiments, thesignal peptide is an IgE HC (Ig heavy chain epsilon-1) signal peptide.

Further provided herein, is a nucleic acid encoding CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine.

Another aspect of the present disclosure provides a CHIKV, DENV, ZIKV,CHIKV/DENV, CHIKV/ZIKV, DENV/ZIKV, or CHIKV/DENV/ZIKV vaccine, whichincludes at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding a signal peptide linked to a CHIKV, DENV,and/or ZIKV antigenic peptide. In some embodiments, the CHIKV, DENV,and/or ZIKV antigenic peptide is a CHIKV, DENV, and/or ZIKV envelopeprotein.

In some embodiments, the signal peptide is a IgE signal peptide. In someembodiments, the signal peptide is an IgE HC (Ig heavy chain epsilon-1)signal peptide. In some embodiments, the signal peptide has the sequenceMDWTWILFLVAAATRVHS (SEQ ID NO: 126). In some embodiments, the signalpeptide is an IgGκ signal peptide. In some embodiments, the signalpeptide has the sequence METPAQLLFLLLLWLPDTTG (SEQ ID NO: 125).

In any of the aspects and embodiments described herein the combinationvaccine is a CHIKV/DENV/ZIKV, CHIKV/DENV, CHIKV/ZIKV, and/or DENV/ZIKVvaccine.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1A shows a schematic depiction of the post-translational process ofCHIKV structural proteins. FIG. 1B shows a schematic depiction of theE1/E2 heterodimer that associates as a trimeric spike on the CHIKV viralsurface.

FIG. 2 shows a phylogenetic tree of chikungunya virus strains derivedfrom complete concatenated open reading frames for the nonstructural andstructural polyproteins. E1 amino acid substitutions that facilitated(Indian Ocean lineage) or prevented (Asian lineage) adaptation to Aedesalbopictus are shown on the right. CAR: Central African republic; ECSA:East/Central/South Africa.

FIG. 3 shows CHIKV envelope protein detection of lysate in HeLa cells 16hours post-transfection.

FIG. 4A is a graph showing the survival rates of AG129 mice vaccinatedwith a single 2 μg dose or two 2 μg doses of Chikungunya E1 antigenadministered either intramuscularly or intradermally. FIG. 4B is a graphshowing the percent weight loss of AG129 mice vaccinated with a single 2μg dose or two 2 μg doses of Chikungunya E1 antigen administered eitherintramuscularly or intradermally. FIG. 4C is a graph showing the healthscores of AG129 mice vaccinated with a single 2 μg dose or two 2 μgdoses of Chikungunya E1 antigen administered either intramuscularly orintradermally.

FIG. 5A is a graph showing the survival rates of AG129 mice vaccinatedwith a single 2 μg dose or two 2 μg doses of Chikungunya E2 antigenadministered either intramuscularly or intradermally. FIG. 5B is a graphshowing the percent weight loss of AG129 mice vaccinated with a single 2μg dose or two 2 μg doses of Chikungunya E2 antigen administered eitherintramuscularly or intradermally. FIG. 5C is a graph showing the healthscores of AG129 mice vaccinated with a single 2 μg dose or two 2 μgdoses of Chikungunya E2 antigen administered either intramuscularly orintradermally.

FIG. 6A is a graph showing the survival rates of AG129 mice vaccinatedwith a single 2 μg dose or two 2 μg doses of Chikungunya C-E3-E2-6K-E1antigen administered either intramuscularly or intradermally. FIG. 6B isa graph showing the percent weight loss of AG129 mice vaccinated with asingle 2 μg dose or two 2 μg doses of Chikungunya C-E3-E2-6K-E1 antigenadministered either intramuscularly or intradermally. FIG. 6C is a graphshowing the health scores of AG129 mice vaccinated with a single 2 μgdose or two 2 μg doses of Chikungunya C-E3-E2-6K-E1 antigen administeredeither intramuscularly or intradermally.

FIG. 7 shows the study design, schedule of injection/bleeding, readout,and survival data for the 2 μg dose study of the CHIKV E1, CHIKV E2, andCHIKV E1/E2/E3/6K/C vaccines.

FIG. 8A is a graph showing the survival rates of AG129 mice vaccinatedwith a single 10 μg dose or two 10 μg doses of Chikungunya E1 antigenadministered either intramuscularly or intradermally. FIG. 8B is a graphshowing the percent weight loss of AG129 mice vaccinated with a single10 μg dose or two 10 μg doses of Chikungunya E1 antigen administeredeither intramuscularly or intradermally. FIG. 8C is a graph showing thehealth scores of AG129 mice vaccinated with a single 10 μg dose or two10 μg doses of Chikungunya E1 antigen administered eitherintramuscularly or intradermally.

FIG. 9A is a graph showing the survival rates of AG129 mice vaccinatedwith a single 10 μg dose or two 10 μg doses of Chikungunya E2 antigenadministered either intramuscularly or intradermally. FIG. 9B is a graphshowing the percent weight loss of AG129 mice vaccinated with a single10 μg dose or two 10 μg doses of Chikungunya E2 antigen administeredeither intramuscularly or intradermally. FIG. 9C is a graph showing thehealth scores of AG129 mice vaccinated with a single 10 μg dose or two10 μg doses of Chikungunya E2 antigen administered eitherintramuscularly or intradermally.

FIG. 10A is a graph showing the survival rates of AG129 mice vaccinatedwith a single 10 μg dose or two 10 μg doses of Chikungunya C-E3-E2-6K-E1antigen administered either intramuscularly or intradermally. FIG. 10Bis a graph showing the percent weight loss of AG129 mice vaccinated witha single 10 μg dose or two 10 μg doses of Chikungunya C-E3-E2-6K-E1antigen administered either intramuscularly or intradermally. FIG. 10Cis a graph showing the health scores of AG129 mice vaccinated with asingle 10 μg dose or two 10 μg doses of Chikungunya C-E3-E2-6K-E1antigen administered either intramuscularly or intradermally.

FIG. 11 shows the study design, schedule of injection/bleeding, readout,and survival data for the 10 μg dose study of the CHIKV E1, CHIKV E2,and CHIKV C-E3-E2-6K-E1 vaccines.

FIG. 12 shows the results of an in vitro transfection of mRNA encodedCHIKV structural proteins. Protein detection in HeLa cell lysate 16 hpost transfection is detected.

FIGS. 13A and 13B are schematics of an exemplary DENV peptide epitope.The polypeptide of FIG. 13A includes two or more epitopes. The epitopescan be of the same sequence or different sequence and can be all T-cellepitopes, all B-cell epitopes or a combination of both. The schematic ofFIG. 13B shows the peptide epitope with various end units for enhancingMHC processing of the peptides.

FIG. 14 is a schematic of a dengue viral genome including structural andnonstructural components.

FIG. 15 shows exemplary dengue peptide epitopes identified using adatabase screen.

FIGS. 16A-16C show Dengue Virus MHC I T cell epitopes.H

FIGS. 17A-17C show Dengue Virus MHC II T cell epitopes.

FIG. 18 is a graph depicting the results of an ELISPOT assay ofdengue-specific peptides.

FIG. 19 is a graph depicting the results of an ELISPOT assay ofdengue-specific peptides.

FIG. 20 is a schematic of a bone marrow/liver/thymus (BLT) mouse anddata on human CD8 T cells stimulated with Dengue peptide epitope.

FIG. 21 shows DENV MHC-1_V5 concatemer transfection in HeLa cells.Triple immunofluorescence using Mitotracker Red (mitochondria), anti-V5,and anti-MHC-1 antibodies plus DAPI was performed. The arrows indicateV5-MHC1 colocalization (bottom right).

FIG. 22 shows DENV MHC-1_V5 concatemer transfection in HeLa cells.Triple immunofluorescence using Mitotracker Red (mitochondria), anti-V5,and anti-MHC-1 antibodies plus DAPI was performed. The arrows indicateregions where V5 preferentially colocalizes with MHC1 and not withMitotracker.

FIG. 23 shows DENV MHC-1_V5 concatemer transfection in HeLa cells.Triple immunofluorescence using Mitotracker Red (mitochondria), anti-V5,and anti-MHC-1 antibodies plus Dapi was performed. V5 has homogeneouscytoplasmic distribution preferentially colocalizes with MHC1 and notwith Mitotracker.

FIGS. 24A and 24B shows the results of an Intracellular CytokineStaining assay performed in PBMC cells.

FIG. 25 shows a schematic of a genomic polyprotein obtained from Zikavirus, Flaviridiaie. The ZIKV genome encodes a polyprotein with threestructural proteins (capsid (C), premembrane/membrane (prM), andenvelope (E, a glycosylation motif previously associated withvirulence)), and seven nonstructural proteins (NS1, NS2A, NS2B, NS3,NS4A, NS4B and NS5). The polyprotein may be cleaved by several hostpeptidase or proteases to generate structural or functional proteins forthe virus. The respective cleavage sites of the peptidases or proteasesare indicated by arrows.

FIG. 26A shows a schematic of a ZIKV vaccine that comprises a RNApolynucleotide encoding a signal peptide fused to Zika prM protein fusedto Zika E protein. FIG. 26B shows a schematic of a ZIKV vaccine thatcomprises a RNA polynucleotide encoding a signal peptide fused to Zika Eprotein. The cleavage junction is located between the signal peptide andthe Zika prM protein and is conserved between Dengue and Zika.

FIG. 27 shows a sequence alignment of currently circulating Zika Virusstrains.

FIG. 28 shows fluorescent staining of non-reduced mammalian celllysates. Tube 1 contains lysed cell precipitate obtained from 293T cellstransfected with ZIKV prME mRNA and stained with secondary antibody only(negative control). Tube 2 contains lysed cell precipitate obtained fromuntransfected 293T cells and stained with anti-ZIKV human serum (1:20)and goat anti-human Alexa Fluor 647 (negative control). Tube 3 containslysed cell precipitate obtained from 293T cells transfected with ZIKVprME mRNA and stained with anti-ZIKV human serum (1:20) and goatanti-human Alexa Fluor 647. Tube 4 contains lysed cell precipitateobtained from 293T cells transfected with ZIKV prME mRNA and stainedwith anti-ZIKV human serum (1:200) and goat anti-human Alexa Fluor 647.The antibodies in anti-ZIKV human serum can detect non-reduced proteinsexpressed by prME mRNA constructs.

FIG. 29 shows a histogram indicating intracellular detection of ZIKAprME protein using human anti-ZIKV serum.

FIGS. 30A-30B show the results of detecting prME protein expression inmammalian cells with fluorescence-activated cell sorting (FACS) using aflow cytometer. Cells expressing prME showed higher fluorescenceintensity when stained with anti-ZIKV human serum.

FIG. 31 shows a graph of neutralizing titers from Balb/c mice immunizedwith ZIKV mRNA vaccine encoding prME.

FIG. 32 shows negative stain images for Hela samples.

FIG. 33A shows a reducing SDS-PAGE gel of Zika VLP. FIG. 33B shows agraph of neutralizing titers obtained from Balb/c mice immunized with aZIKV mRNA vaccine.

FIG. 34A shows FACS analyses of cells expressing DENV2 prMEs usingdifferent antibodies against Dengue envelope protein. Numbers in theupper right corner of each plot indicate mean fluorescent intensity.FIG. 34B shows a repeat of staining in triplicate and in two differentcell lines (HeLa and 293T).

FIG. 35 shows an in vitro antigen presentation assays using OVA (peptideepitope of ovalbumin) multitopes to test different DENV mRNA vaccineconstruct configurations.

FIG. 36 is a graph showing the kinetics of OVA peptide presentation inJawsii cells. All mRNAs tested are formulated in MC3 lipidnanoparticles.

FIG. 37 is a graph showing the Mean Fluorescent Intensity (MFI) ofantibody binding to DENV-1, 2, 3, and 4 prME epitopes presented on thecell surface.

FIGS. 38A-38D are graphs showing the design and the results of achallenge study in AG129 mice. FIG. 38A shows the immunization,challenge, and serum collection schedules.

FIG. 38B shows the survival of the AG129 mice challenged with DengueD2Y98P virus after being immunized with the indicated DENV mRNAvaccines. All immunized mice survived 11 days post infection, while theunimmunized (control) mice died. FIGS. 38C and 38D show the weight lossof the AG129 mice post infection. Vaccine 1, 7, 8, or 9 correspond toDENV vaccine construct 22, 21, 23, or 24 of the present disclosure,respectively.

FIG. 39 is a graph showing the results of an in vitro neutralizationassay using serum from mice immunized with the DENV mRNA vaccines inFIGS. 39A-39D.

FIGS. 40A-40I are graphs showing the results of a challenge study inAG129 mice. The challenge study design is shown in Table 40. FIGS.40A-40F show the survival, weight loss, and heath score of the AG129mice challenged with D2Y98P virus after being immunized with the DENVmRNA vaccine groups 1-12 in Table 40. FIGS. 40G-40I show the survival,weight loss, and heath score of the AG129 mice challenged with D2Y98Pvirus after being immunized with the DENV mRNA vaccine groups 13-19 inTable 40.

FIG. 41 is a negative-stain electron microscopy image of the virus-likeparticles (VLPs) assembled from the antigens (prME) encoded by the DENVmRNA vaccines. DENV mRNA vaccine constructs 21-24 in Table 38 weretested. Construct 23 is the vaccine construct used by Sanofi in its DENVvaccines. Constructs 21, 22, and 24 produced more uniform VLPs,suggesting that these VLPs may be more superior in their immunogenicitythan the VLPs produced from construct 23.

FIGS. 42A-42B are graphs showing the survival curves from a CHIKVchallenge study in AG129 mice immunized with CHIKV mRNA vaccines in 10μg, 2 μg, or 0.04 μg doses. Mice were divided into 14 groups (1-4 and7-16, n=5). FIG. 42A shows the survival curve of mice groups 1-4 and 7-9challenged on day 56 post immunization. FIG. 42B shows the survivalcurve of mice groups 10-16 challenged on day 112 post immunization.Survival curves were plotted as “percent survival” versus “days postinfection.” See also Table 45 for survival percentage.

FIGS. 43A-43B are graphs showing the weight changes post challenge inAG129 mice immunized with CHIKV mRNA vaccines. FIG. 43A shows the weightchange of mice groups 1-4 and 7-9 challenged on day 56 postimmunization. FIG. 43B shows the weight changes of mice groups 10-16challenged on day 112 post immunization. Initial weights were assessedon individual mice on study Day 0 and daily thereafter. The mean percentweights for each group compared to their percent weight on Day 0(baseline) were plotted against “days post-infection”. Error barsrepresent the standard deviation (SD).

FIGS. 44A-44B are graphs showing the post challenge heath scores ofAG129 mice immunized with CHIKV mRNA vaccines. FIG. 44A shows the healthscores of mice groups 1-4 and 7-9 challenged on day 56 postimmunization. FIG. 44B shows the health score of mice groups 10-16challenged on day 112 post immunization. The mean health scores for eachgroup were plotted against “days post infection” and error barsrepresent the SD. Mean health scores were calculated based onobservations described in Table 5.

FIGS. 45A-45C are graphs showing the antibody titers measured by ELISAassays in the serum of AG129 mice (groups 1-4 and 7-9) 28 days postimmunization with CHIKV mRNA vaccines. FIG. 45A shows the serum antibodytiters against CHIKV E1 protein. FIG. 45B shows the serum antibodytiters against CHIKV E2 protein. FIG. 45C shows the serum antibodytiters against CHIKV lysate.

FIGS. 46A-46C are graphs showing the antibody titers measured by ELISAassays in the serum of AG129 mice (groups 10-16) 28 days postimmunization with CHIKV mRNA vaccine. FIG. 45A shows the serum antibodytiters against CHIKV E1 protein. FIG. 46B shows the serum antibodytiters against CHIKV E2 protein. FIG. 46C shows the serum antibodytiters against CHIKV lysate.

FIGS. 47A-47C are graphs showing the antibody titers measured by ELISAassays in the serum of AG129 mice (groups 10-16) 56 days postimmunization with CHIKV mRNA vaccine. FIG. 47A shows the serum antibodytiters against CHIKV E1 protein. FIG. 47B shows the serum antibodytiters against CHIKV E2 protein. FIG. 47C shows the serum antibodytiters against CHIKV lysate.

FIGS. 48A-48C are graphs showing the antibody titers measured by ELISAassays in the serum of AG129 mice (groups 10-16) 112 days postimmunization with CHIKV mRNA vaccine. FIG. 48A shows the serum antibodytiters against CHIKV E1 protein. FIG. 48B shows the serum antibodytiters against CHIKV E2 protein. FIG. 48C shows the serum antibodytiters against CHIKV lysate.

FIG. 49 shows different antigens based on the Chikungunya structuralprotein from three different genotypes.

FIG. 50 shows a set of graphs depicting results of an ELISA assay toidentify the amount of antibodies produced in AG129 mice in response tovaccination with mRNA encoding secreted CHIKV E1 structural protein,secreted CHIKV E2 structural protein, or CHIKV full structuralpolyprotein C-E3-E2-6k-E1 at a dose of 10 μg or 2 μg at 28 days postimmunization.

FIG. 51 shows a set of graphs depicting results of an ELISA assay toidentify the amount of antibodies produced in AG129 mice in response tovaccination with mRNA encoding secreted CHIKV E1 structural protein,secreted CHIKV E2 structural protein, or CHIKV full structuralpolyprotein C-E3-E2-6k-E1 at a dose of 10 μg or 2 μg at 28 days postimmunization. The two panels depict different studies.

FIG. 52 is a graph depicting comparison of ELISA titers from the data ofFIG. 50 to survival in the data of FIG. 51 left panel.

FIG. 53 shows a set of graphs depicting efficacy results in mice inresponse to vaccination with mRNA encoding CHIKV full structuralpolyprotein C-E3-E2-6k-E1 at a dose of 10 μg (left panels), 2 μg (middlepanels) or 0.4 μg (right panels) at 56 days (top panels) or 112 days(bottom panels) post immunization.

FIG. 54 shows a set of graphs depicting amount of neutralizing antibodyproduced in mice in response to vaccination with mRNA encoding CHIKVfull structural polyprotein C-E3-E2-6k-E1 at a dose of 10 μg, 2 μg, or0.4 μg at 56 days post immunization.

FIG. 55 shows a set of graphs depicting binding antibody produced inmice in response to vaccination with mRNA encoding CHIKV full structuralpolyprotein C-E3-E2-6k-E1 at a dose of 10 μg, 2 μg, or 0.4 μg at 56 dayspost immunization (top panels) and the corresponding correlation betweenbinding and neutralizing antibodies (bottom panels).

FIG. 56 shows a set of graphs depicting amount of neutralizing antibodyproduced in A129 mice in response to vaccination with mRNA encodingCHIKV full structural polyprotein C-E3-E2-6k-E1 at a dose of 10 μg, 2μg, or 0.4 μg at 56 days post immunization against three differentstrains of CHIKV, African-Senegal (left panel), La Reunion (middlepanel) and CDC CAR (right panel).

FIG. 57 shows a graph depicting neutralizing antibodies against CHIKVS27 strain.

FIG. 58 is a graph depicting antibody titer against CHIKV lysate post3rd vaccination 10 with the mRNA vaccine in Sprague Dawley rats.

FIG. 59 shows a set of graphs depicting antibody titers followingvaccination of mice with mRNA encoded CHIKV polyprotein (C-E3-E2-6K-E1).

FIG. 60 shows a set of plots depicting cytokine secretion and T-cellactivation following vaccination of mice with mRNA encoded CHIKVpolyprotein (C-E3-E2-6K-E1) (SEQ ID NO: 13).

FIGS. 61A-61B show a set of graphs depicting CD8+ T cell activationfollowing vaccination of mice with mRNA encoded CHIKV polyprotein(C-E3-E2-6K-E1) (SEQ ID NO: 13).

DETAILED DESCRIPTION

Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccinesthat are useful for vaccinating against one or multiple viruses. Thevaccines, including combination vaccines, of the invention encodeantigens from chikungunya virus (CHIKV), Zika virus (ZIKV), Dengue virus(DENV), or any combination of two or three of the foregoing viruses. Abalanced immune response, comprising both cellular and humoral immunity,can be generated against CHIKV, against DENV, against ZIKV, againstCHIKV and DENV, against CHIKV and ZIKV, against DENV and ZIKV, oragainst CHIKV, DENV and ZIKV, using the constructs of the inventionwithout many of the risks associated with DNA vaccines and liveattenuated vaccines. The various RNA vaccines disclosed herein producedsurprising efficacy in animal models of Chikungunya infection, andDengue infection, the results of which are discussed in detail in theExamples section. Specifically, RNA polynucleotide vaccines having anopen reading frame encoding for a variety of Chikungunya antigensproduced significant immunity, whereas traditional Chikungunya vaccineshave not (e.g. attenuated chikungunya viruses). The CHIKV RNApolynucleotide vaccines disclosed herein encoding either CHIKV-E1,CHIKV-E2 or CHIKV-C-E3-E2-6K-E1 demonstrated a survival rate of 60%-100%after two administrations. Specifically, two injections of CHIKV E1 mRNAvaccine provided nearly full protection against infection whenadministered intramuscularly (IM) (60% survival) or intradermally (ID)(80% survival). Two injections of CHIKV E2 mRNA vaccine or CHIKVC-E3-E2-6K-E1 vaccine provided full protection (100% survival) againstinfection when administered via IM or ID. Importantly, a singleinjection (no booster dose) of CHIKV C-E3-E2-6K-E1 vaccine provided fullprotection (100% survival) against infection when administered via IM orID.

DENV RNA vaccines and ZIKV vaccines are also disclosed herein as well ascombination DENV and CHIKV, CHIKV and ZIKV, and DENV and ZIKV vaccines.The combination vaccines of CHIKV, DENV and ZIKV, DENV and ZIKV, CHIKVand ZIKV, or CHIKV and DENV can provide a means for protecting againsttwo or more viral infections in a single vaccine.

Chikungunya virus is a small (about 60-70 nm-diameter), spherical,enveloped, positive-strand RNA virus having a capsid with icosahedralsymmetry. The virion consists of an envelope and a nucleocapsid. Thevirion RNA is infectious and serves as both genome and viral messengerRNA. The genome is a linear, ssRNA(+) genome of 11,805 nucleotides whichencodes for two polyproteins that are processed by host and viralproteases into non-structural proteins (nsP1, nsP2, nsP3, and RdRpnsP4)necessary for RNA synthesis (replication and transcription) andstructural proteins (capsid and envelope proteins C, E3, E2, 6K, and E1)which attach to host receptors and mediate endocytosis of virus into thehost cell. (FIG. 1). The E1 and E2 glycoproteins form heterodimers thatassociate as 80 trimeric spikes on the viral surface covering thesurface evenly. The envelope glycoproteins play a role in attachment tocells. The capsid protein possesses a protease activity that results inits self-cleavage from the nascent structural protein. Following itscleavage, the capsid protein binds to viral RNA and rapidly assemblesinto icosahedric core particles. The resulting nucleocapsid eventuallyassociates with the cytoplasmic domain of E2 at the cell membrane,leading to budding and formation of mature virions.

E2 is an envelope glycoprotein responsible for viral attachment totarget host cell, by binding to the cell receptor. E2 is synthesized asa p62 precursor which is processed at the cell membrane prior to virionbudding, giving rise to an E2-E1 heterodimer. The C-terminus of E2 isinvolved in budding by interacting with capsid proteins.

E1 is an envelope glycoprotein with fusion activity, which fusionactivity is inactive as long as E1 is bound to E2 in the mature virion.Following virus attachment to target cell and endocytosis, acidificationof the endosome induces dissociation of the E1/E2 heterodimer andconcomitant trimerization of the E1 subunits. The E1 trimer is fusionactive and promotes the release of the viral nucleocapsid in thecytoplasm after endosome and viral membrane fusion.

E3 is an accessory protein that functions as a membranetranslocation/transport signal for E1 and E2.

6K is another accessory protein involved in virus glycoproteinprocessing, cell permeabilization, and the budding of viral particlesLike E3, it functions as a membrane transport signal for E1 and E2.

The CHIKV structural proteins have been shown to be antigenic, whichproteins, fragments, and epitopes thereof are encompassed within theinvention. A phylogenetic tree of Chikungunya virus strains derived fromcomplete concatenated open reading frames for the nonstructural andstructural polyproteins shows key envelope glycoprotein E1 amino acidsubstitutions that facilitated (Indian Ocean lineage) or prevented(Asian lineage) adaptation to Aedes albopictus. There are membrane-boundand secreted forms of E1 and E2, as well as the full length polyproteinantigen (C-E3-E2-6K-E1), which retains the protein's nativeconformation. Additionally, the different Chikungunya genotypes, strainsand isolates can also yield different antigens, which are functional inthe constructs of the invention. For example, there are severaldifferent Chikungunya genotypes: Indian Ocean, East/Central/SouthAfrican (ECSA), Asian, West African, and the Brazilian isolates(ECSA/Asian). There are three main Chikungunya genotype. These are ESCA(East-South-Central Africa); Asia; and West Africa. While sometimesnames differ in publications all belong to these three geographicalstrains.

Dengue virus is a mosquito-borne (Aedes aegypti/Aedes albopictus) memberof the family Flaviviridae (positive-sense, single-stranded RNA virus).The dengue virus genome encodes ten genes and is translated as a singlepolypeptide which is cut into ten proteins: the capsid, envelope,membrane, and nonstructural proteins (NS1, NS2A, NS2B, NS3, SN4A, NS4B,and NS5 proteins). The virus' main antigen is DENe, which is a componentof the viral surface and is thought to facilitate the binding of thevirus to cellular receptors (Heinz et al., Virology. 1983, 126:525).There are four similar but distinct serotypes of dengue virus (DEN-1,DEN-2, DEN-3, and DEN-4), which result annually in an estimated 50-100million cases of dengue fever and 500,000 cases of the more severedengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (Gubler et al.,Adv Virus Res. 1999, 53:35-70). The four serotypes show immunologicalcross-reactivity, but are distinguishable in plaque reductionneutralization tests and by their respective monoclonal antibodies. Thedengue virus E protein includes a serotype-specific antigenicdeterminant and determinants necessary for virus neutralization (Masonet al., J Gen Virol. 1990, 71:2107-2114).

After inoculation, the dendritic cells become infected and travel tolymph nodes. Monocytes and macrophages are also targeted shortlythereafter. Generally, the infected individual will be protected againsthomotypic reinfection for life; however, the individual will only beprotected against other serotypes for a few weeks or months (Sabin, Am JTrop Med Hyg. 1952, 1:30-50). In fact, DHF/DSS is generally found inchildren and adults infected with a dengue virus serotype differing fromtheir respective primary infection. Thus, it is necessary to develop avaccine that provides immunity to all four serotypes.

Along with other viruses in the Flaviviridae family, Zika virus isenveloped and icosahedral with a non-segmented, single-stranded,positive sense RNA genome. It is most closely related to the Spondwenivirus and is one of the two viruses in the Spondweni virus Glade. Thevirus was first isolated in 1947 from a rhesus monkey in the Zika Forestof Uganda, Africa and was isolated for the first time from humans in1968 in Nigeria. From 1951 through 1981, evidence of human infection wasreported from other African countries such as Uganda, Tanzania, Egypt,Central African Republic, Sierra Leone and Gabon, as well as in parts ofAsia including India, Malaysia, the Philippines, Thailand, Vietnam andIndonesia. It is transmitted by mosquitoes and has been isolated from anumber of species in the genus Aedes—Aedes aegypti, Aedes africanus,Aedes apicoargenteus, Aedes furcifer, Aedes luteocephalus and Aedesvitattus. Studies show that the extrinsic incubation period inmosquitoes is about 10 days. The vertebrate hosts of the virus includemonkeys and humans.

As of early 2016, the most widespread outbreak of Zika fever, caused bythe Zika virus, is ongoing primarily in the Americas. The outbreak beganin April 2015 in Brazil, and subsequently spread to other countries inSouth America, Central America, and the Caribbean.

The Zika virus was first linked with newborn microcephaly during theBrazil Zika virus outbreak. In 2015, there were 2,782 cases ofmicrocephaly compared with 147 in 2014 and 167 in 2013. The BrazilianHealth Ministry has reported 4783 cases of suspected microcephaly as ofJanuary 30, an increase of more than 1000 cases from a week earlier.Confirmation of many of the recent cases is pending, and it is difficultto estimate how many cases went unreported before the recent awarenessof the risk of virus infections.

What is important is not only the number of cases but also the clinicalmanifestation of the cases. Brazil is seeing severe cases ofmicrocephaly, which are more likely to be paired with greaterdevelopmental delays. Most of what is being reported out of Brazil ismicrocephaly with other associated abnormalities. The potentialconsequence of this is the fact that there are likely to be subclinicalcases where the neurological sequelae will only become evident as thechildren grow.

Zika virus has also been associated with an increase in a rare conditionknown as Guillain-Barré, where the infected individual becomesessentially paralyzed. During the Zika virus outbreak in FrenchPolynesia, 74 patients which had had Zika symptoms—out of them, 42 werediagnosed as Guillain-Barré syndrome. In Brazil, 121 cases ofneurological manifestations and Guillain-Barré syndrome (GBS) werereported, all cases with a history of Zika-like symptoms.

In some embodiments, ZIKV vaccines comprise RNA (e.g., mRNA) encoding aZIKV antigenic polypeptide having at least 95%, at least 96%, at least97%, at least 98% or at least 99% identity with ZIKV polyprotein andhaving ZIKV polyprotein activity, respectively. The ZIKV polyprotein iscleaved into capsid, precursor membrane, envelope, and non-structuralproteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).

A protein is considered to have ZIKV polyprotein activity if, forexample, it facilitates the attachment of the viral envelope to hostreceptors, mediates internalization into the host cell, and aids infusion of the virus membrane with the host's endosomal membrane.

The RNA vaccines may be utilized in various settings depending on theprevalence of the infection or the degree or level of unmet medicalneed. The RNA vaccines may be utilized to treat and/or prevent a CHIKV,DENV and/or ZIKV infection of various genotypes, strains, and isolates.The RNA vaccines have superior properties in that they produce muchlarger antibody titers and produce responses early than commerciallyavailable anti-viral therapeutic treatments. While not wishing to bebound by theory, it is believed that the RNA vaccines, as mRNApolynucleotides, are better designed to produce the appropriate proteinconformation upon translation as the RNA vaccines co-opt naturalcellular machinery. Unlike traditional vaccines which are manufacturedex vivo and may trigger unwanted cellular responses, the RNA vaccinesare presented to the cellular system in a more native fashion.

The entire contents of International Application No. PCT/US2015/02740 isincorporated herein by reference.

Nucleic Acids/Polynucleotides

Vaccines, including combination vaccines, as provided herein, compriseat least one (one or more) ribonucleic acid (RNA) polynucleotide havingan open reading frame encoding at least one CHIKV antigenic polypeptide,at least one ZIKV antigenic polypeptide, at least one DENV antigenicpolypeptide, at least one CHIKV antigenic polypeptide and at least oneDENV antigenic polypeptide, at least one CHIKV antigenic polypeptide andat least one ZIKV antigenic polypeptide, at least one ZIKV antigenicpolypeptide and at least one DENV antigenic polypeptide, or at least oneCHIKV antigenic polypeptide, at least one DENV antigenic polypeptide andat least one ZIKV antigenic polypeptide. In some embodiments, thevaccine, including combination vaccines, comprise at least one RNApolynucleotide, e.g., mRNA, having an open reading frame encoding two ormore different CHIKV antigenic polypeptides, ZIKV antigenicpolypeptides, and/or DENV antigenic polypeptides (e.g., two, three,four, five or more different antigenic polypeptides). In someembodiments, the combination vaccine comprises at least one RNApolynucleotide having an open reading frame encoding a CHIKV antigenicpolypeptide or epitope, a ZIKV antigenic polypeptide or epitope, a DENVantigenic polypeptide or epitope, or a combination of any two or threeof the forgoing. The term “nucleic acid,” in its broadest sense,includes any compound and/or substance that comprises a polymer ofnucleotides. These polymers are referred to as polynucleotides. As usedherein the term polypeptide refers to full-length proteins, proteinfragments, variants, and epitopes.

In some embodiments, an RNA polynucleotide, e.g., mRNA, of a combinationvaccine encodes at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 antigenicpolypeptides. In some embodiments, an RNA polynucleotide comprises 30 to12,000 or more nucleotides. For example, a polynucleotide may include 30to 100, 101 to 200, 200 to 500, 200 to 1000, 200 to 1500, 200 to 2000,200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, 1500 to 4000, 1500 to5000, 2000 to 3000, 2000 to 4000, 2000 to 5000, 5000 to 7500, 7500 to10,000, or 10,000 to 12,000 nucleotides.

In some embodiments, the combination vaccine comprises at least one RNApolynucleotide having an open reading frame encoding a Chikungunyastructural protein or an antigenic fragment or an antigenic epitopethereof. In some embodiments, the RNA polynucleotide has an open readingframe encoding a Chikungunya envelope and/or capsid antigenicpolypeptide selected from a CHIKV E1, E2, E3, 6K, and capsid (C)antigenic polypeptide. In some embodiments, the RNA polynucleotide hasan open reading frame encoding any combination of CHIKV E1, E2, E3, 6K,and capsid (C) antigenic polypeptides, for example, a combinationselected from CHIKV E1 and E2 antigens, CHIKV E1 and E3 antigens, CHIKVE2 and E3 antigens, CHIKV E1, E2, and E3 antigens, CHIKV E1, E2, E3, andC antigens, CHIKV E1, E2, and 6K antigens, CHIKV E2, E3 and 6K antigens,CHIKV E1, E3, and 6K antigens, and CHIKV E1, E2, E3, 6K, and C antigens.

Some embodiments of the present disclosure provide DENV vaccines thatinclude at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one DENV antigenic polypeptide oran immunogenic fragment or epitope thereof. Some embodiments of thepresent disclosure provide DENV vaccines that include at least one RNApolynucleotide having an open reading frame encoding two or more DENVantigenic polypeptides or an immunogenic fragment or epitope thereof.Some embodiments of the present disclosure provide DENV vaccines thatinclude two or more RNA polynucleotides having an open reading frameencoding two or more DENV antigenic polypeptides or immunogenicfragments or epitopes thereof. The one or more DENV antigenicpolypeptides may be encoded on a single RNA polynucleotide or may beencoded individually on multiple (e.g., two or more) RNApolynucleotides.

In some embodiments, the at least one RNA polynucleotide may encode atleast one DENV antigenic polypeptide. In some instances the dengue viralantigenic polypeptide is an intact dengue virus peptide or other largeantigen (i.e. greater than 25 amino acids in length). In someembodiments, the at least one RNA polynucleotide encodes a DENV capsidprotein or immunogenic fragment or epitope thereof. In some embodiments,the at least one RNA polynucleotide encodes a DENV envelope protein orimmunogenic fragment or epitope thereof. In some embodiments, the atleast one RNA polynucleotide encodes a DENV membrane protein orimmunogenic fragment or epitope thereof. In some embodiments, the atleast one RNA polynucleotide encodes a DENV nonstructural protein orimmunogenic fragment or epitope thereof. Large gene segments innon-structural genes, in particular may be used for antigens. In someembodiments, the DENV non-structural protein is selected from NS1, NS2A,NS2B, NS3, SN4A, NS4B, and NS5 proteins, or immunogenic fragments orepitopes thereof. In some embodiments, the DENV non-structural proteinis NS3. In some embodiments, the at least one RNA polynucleotide encodesDENe, which is a component of the viral surface and is thought tofacilitate the binding of the virus to cellular receptors. In any ofthese embodiments, the at least one RNA polynucleotide encodes a DENVpolypeptide from a DENV serotype selected from DENV-1, DENV-2, DENV-3,and DENV-4. For example, the DENV polypeptide may be one or morepolypeptides encoded by SEQ ID NO: 15 (DENV1), SEQ ID NO: 17 (DENV2),SEQ ID NO: 19 (DENV3), and SEQ ID NO: 21 (DENV4), In some embodiments,the DENV polypeptide is a polypeptide found in SEQ ID NO: 14 (DENV1),SEQ ID NO: 16 (DENV2), SEQ ID NO: 18 DENV3), and/or SEQ ID NO: 20(DENV4). In some embodiments, the Dengue virus (DENV) vaccine comprisesat least one (one or more) ribonucleic acid (RNA) polynucleotide havingan open reading frame encoding SEQ ID NO: 23 or an immunogenic fragmentor epitope thereof. In some embodiments, the Dengue virus (DENV) vaccinecomprises at least one (one or more) ribonucleic acid (RNA)polynucleotide having an open reading frame encoding SEQ ID NO: 26 or animmunogenic fragment or epitope thereof. In some embodiments, the Denguevirus (DENV) vaccine comprises at least one (one or more) ribonucleicacid (RNA) polynucleotide having an open reading frame encoding SEQ IDNO: 29 or an immunogenic fragment or epitope thereof. In someembodiments, the Dengue virus (DENV) vaccine comprises at least one (oneor more) ribonucleic acid (RNA) polynucleotide having an open readingframe encoding SEQ ID NO: 32 or an immunogenic fragment or epitopethereof. In some embodiments, the DENV RNA polynucleotide comprises SEQID NO: 25 (or is encoded by SEQ ID NO: 24) or a fragment thereof. Insome embodiments, the DENV RNA polynucleotide comprises SEQ ID NO: 28(or is encoded by SEQ ID NO: 27) or a fragment thereof. In someembodiments, the DENV RNA polynucleotide comprises SEQ ID NO: 31 (or isencoded by SEQ ID NO: 30) or a fragment thereof. In some embodiments,the DENV RNA polynucleotide comprises SEQ ID NO: 34 (or is encoded bySEQ ID NO: 33) or a fragment thereof. In some embodiments, the DENV RNApolynucleotide encodes a polypeptide comprising SEQ ID NO:23 or animmunogenic fragment or epitope thereof. In some embodiments, the DENVRNA polynucleotide encodes a polypeptide comprising SEQ ID NO: 26 or animmunogenic fragment or epitope thereof. In some embodiments, the DENVRNA polynucleotide encodes a polypeptide comprising SEQ ID NO: 29 or animmunogenic fragment or epitope thereof. In some embodiments, the DENVRNA polynucleotide encodes a polypeptide comprising SEQ ID NO: 32 or animmunogenic fragment or epitope thereof.

Dengue virus (DENV) vaccine antigens, as provided herein, comprise atleast one (one or more) ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one DENV antigenic polypeptide. Insome embodiments, the DENV antigenic polypeptide is longer than 25 aminoacids and shorter than 50 amino acids. Thus, polypeptides include geneproducts, naturally occurring polypeptides, synthetic polypeptides,homologs, orthologs, paralogs, fragments and other equivalents,variants, and analogs of the foregoing. A polypeptide may be a singlemolecule or may be a multi-molecular complex such as a dimer, trimer ortetramer. Polypeptides may also comprise single chain or multichainpolypeptides such as antibodies or insulin and may be associated orlinked. Most commonly, disulfide linkages are found in multichainpolypeptides. The term polypeptide may also apply to amino acid polymersin which at least one amino acid residue is an artificial chemicalanalogue of a corresponding naturally-occurring amino acid.

In other embodiments the antigen is a concatemeric peptide antigencomposed of multiple peptide epitopes. In some embodiments, a RNApolynucleotide of a DENV vaccine encodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5,2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6,4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8,8-10, 8-9 or 9-10 antigenic polypeptides. In some embodiments, a RNApolynucleotide of a DENV vaccine encodes at least 10, 20, 30, 40, 50,60, 70, 80, 90 or 100 antigenic polypeptides. In some embodiments, a RNApolynucleotide of a DENV vaccine encodes at least 100 or at least 200antigenic polypeptides. In some embodiments, a RNA polynucleotide of aDENV vaccine encodes 1-10, 5-15, 10-20, 15-25, 20-30, 25-35, 30-40,35-45, 40-50, 1-50, 1-100, 2-50 or 2-100 antigenic polypeptides.

In order to design useful epitopes, publically available databases, suchas the Immune Epitope Database (IEDB), may be used to predictimmunogenic Dengue T cell epitopes showing strong homology across all 4Dengue serotypes. For instance, the IEDB is a free database offeringsearching of experimental data characterizing antibody and T cellepitopes and assisting in the prediction and analysis of B cell and Tcell epitopes. The Dengue peptides identified by database may beconfirmed using peptides in MHC allele binding assays (such as thosedescribed in the Examples provided herein) and/or restimulation assaysduring the acute phase of Dengue infection (i.e. Day 7). Some examplesof epitopes are shown in FIG. 15. These epitopes may be evaluated intest mice and using an assay such as that shown in FIG. 18.

Some embodiments of the present disclosure provide ZIKV vaccines,including combination vaccines, that include at least one ribonucleicacid (RNA) polynucleotide having an open reading frame encoding at leastone ZIKV antigenic polypeptide or an immunogenic fragment or epitopethereof. Some embodiments of the present disclosure provide ZIKVcombination vaccines that include at least one RNA polynucleotide havingan open reading frame encoding two or more ZIKV antigenic polypeptidesor an immunogenic fragment or epitope thereof. Some embodiments of thepresent disclosure provide ZIKV vaccines that include two or more RNApolynucleotides having an open reading frame encoding two or more ZIKVantigenic polypeptides or immunogenic fragments or epitopes thereof. Theone or more ZIKV antigenic polypeptides may be encoded on a single RNApolynucleotide or may be encoded individually on multiple (e.g., two ormore) RNA polynucleotides.

In some embodiments, the at least one RNA polynucleotide may encode atleast one ZIKV antigenic polypeptide. In some instances the ZIKVantigenic polypeptide is an intact ZIKV peptide or other large antigen(i.e. greater than 25 amino acids in length). In any of theseembodiments, the at least one RNA polynucleotide encodes a ZIKVpolypeptide from a ZIKV serotype selected from MR 766, SPH2015 orACD75819. For example, the ZIKV polypeptide may be one or morepolypeptides encoded by SEQ ID NO: 67-134 or an immunogenic fragment orepitope thereof.

Zika virus (ZIKV) vaccines, including combination vaccines, as providedherein, comprise at least one (one or more) ribonucleic acid (RNA)polynucleotide having an open reading frame encoding at least one ZIKVantigenic polypeptide. In some embodiments, the ZIKV antigenicpolypeptide is longer than 25 amino acids and shorter than 50 aminoacids. Thus, polypeptides include gene products, naturally occurringpolypeptides, synthetic polypeptides, homologs, orthologs, paralogs,fragments and other equivalents, variants, and analogs of the foregoing.A polypeptide may be a single molecule or may be a multi-molecularcomplex such as a dimer, trimer or tetramer. Polypeptides may alsocomprise single chain or multichain polypeptides such as antibodies orinsulin and may be associated or linked. Most commonly, disulfidelinkages are found in multichain polypeptides. The term polypeptide mayalso apply to amino acid polymers in which at least one amino acidresidue is an artificial chemical analogue of a correspondingnaturally-occurring amino acid.

The generation of antigens that elicit a desired immune response (e.g. Band/or T-cell responses) against targeted polypeptide sequences invaccine development remains a challenging task. The invention involvestechnology that overcome hurdles associated with vaccine development.Through the use of the technology of the invention, it is possible totailor the desired immune response by selecting appropriate T or B cellepitopes which, by virtue of the fact that they are processedintra-cellularly, are able to be presented more effectively on MHC-1 orMHC-2 molecules (depending on whether they are T or B-cell epitope,respectively). In particular, the invention involves the generation ofDENV concatemers of epitopes (particularly T cell epitopes) preferablyinterspersed with cleavage sites by proteases that are abundant inAntigen Presenting Cells (APCs). These methods mimic antigen processingand may lead to a more effective antigen presentation than can beachieved with peptide antigens.

The fact that the peptide epitopes of the invention are expressed fromRNA as intracellular peptides provides advantages over prior artpeptides that are delivered as exogenous peptides or as DNA. The RNA isdelivered intra-cellularly and expresses the epitopes in proximity tothe appropriate cellular machinery for processing the epitopes such thatthey will be recognized by the appropriate immune cells. Additionally, atargeting sequence will allow more specificity in the delivery of thepeptide epitopes.

In some embodiments the DENV mRNA vaccine of the invention is apoly-epitopic RNA. Poly-epitopes consist of strings of epitopes on thesame mRNA. The RNA sequences that code for the peptide epitopes may beinterspersed by sequences that code for amino acid sequences recognizedby proteolytic enzymes, by other linkers or linked directly.

A concatemeric peptide as used herein is a series of at least twopeptide epitopes linked together to form the propeptide. In someembodiments a concatemeric peptide is composed of 3 or more, 4 or more,5 or more 6 or more 7 or more, 8 or more, 9 or more peptide epitopes. Inother embodiments the concatemeric peptide is composed of 1000 or less,900 or less, 500 or less, 100 or less, 75 or less, 50 or less, 40 orless, 30 or less, 20 or less or 100 or less peptide epitopes. In yetother embodiments a concatemeric peptide has 3-100, 5-100, 10-100,15-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 55-100,60-100, 65-100, 70-100, 75-100, 80-100, 90-100, 5-50, 10-50, 15-50,20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 100-150, 100-200, 100-300,100-400, 100-500, 50-500, 50-800, 50-1,000, or 100-1,000 peptideepitopes.

An epitope, also known as an antigenic determinant, as used herein is aportion of an antigen that is recognized by the immune system in theappropriate context, specifically by antibodies, B cells, or T cells.Epitopes include B cell epitopes and T cell epitopes. B-cell epitopesare peptide sequences which are required for recognition by specificantibody producing B-cells. B cell epitopes refer to a specific regionof the antigen that is recognized by an antibody. The portion of anantibody that binds to the epitope is called a paratope. An epitope maybe a conformational epitope or a linear epitope, based on the structureand interaction with the paratope. A linear, or continuous, epitope isdefined by the primary amino acid sequence of a particular region of aprotein. The sequences that interact with the antibody are situated nextto each other sequentially on the protein, and the epitope can usuallybe mimicked by a single peptide. Conformational epitopes are epitopesthat are defined by the conformational structure of the native protein.These epitopes may be continuous or discontinuous, i.e. components ofthe epitope can be situated on disparate parts of the protein, which arebrought close to each other in the folded native protein structure.

T-cell epitopes are peptide sequences which, in association withproteins on APC, are required for recognition by specific T-cells. Tcell epitopes are processed intracellularly and presented on the surfaceof APCs, where they are bound to MHC molecules including MHC class IIand MHC class I.

The present disclosure, in some aspects, relates to a process ofdeveloping T or B cell concatemeric epitopes or concatemeric epitopescomposed of both B and T cell epitopes. Several tools exist foridentifying various peptide epitopes. For instance, epitopes can beidentified using a free or commercial database (Lonza Epibase, antitopefor example). Such tools are useful for predicting the most immunogenicepitopes within a target antigen protein. The selected peptides may thenbe synthesized and screened in human HLA panels, and the mostimmunogenic sequences are used to construct the mRNA polynucleotidesencoding the concatemeric antigens. One strategy for mapping epitopes ofCytotoxic T-Cells based on generating equimolar mixtures of the fourC-terminal peptides for each nominal 11-mer across your an protein. Thisstrategy would produce a library antigen containing all the possibleactive CTL epitopes.

The peptide epitope may be any length that is reasonable for an epitope.In some embodiments the peptide epitope is 9-30 amino acids. In otherembodiments the length is 9-22, 9-29, 9-28, 9-27, 9-26, 9-25, 9-24,9-23, 9-21, 9-20, 9-19, 9-18, 10-22, 10-21, 10-20, 11-22, 22-21, 11-20,12-22, 12-21, 12-20, 13-22, 13-21, 13-20, 14-19, 15-18, or 16-17 aminoacids. In some embodiments, the optimal length of a peptide epitope maybe obtained through the following procedure: synthesizing a V5 tagconcatemer-test protease site, introducing it into DC cells (forexample, using an RNA Squeeze procedure, lysing the cells, and thenrunning an anti-V5 Western blot to assess the cleavage at proteasesites.

In some embodiments, the RNA polynucleotide of the combination vaccineis encoded by at least one nucleic acid sequence selected from SEQ IDNO: 1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34,144-152 or 199-212 (DENV), and 48-66 (ZIKV). In some embodiments, theRNA polynucleotide of the combination vaccine is encoded by at least onefragment of a nucleic acid sequence selected from SEQ ID NO: 1-13(CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or199-212 (DENV), and 48-66 (ZIKV). In some embodiments, the RNApolynucleotide of the combination vaccine is encoded by at least oneepitope sequence of a nucleic acid sequence selected from SEQ ID NO:1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or199-212 (DENV), or 48-66 (ZIKV).

In particular embodiments, the RNA polynucleotide is encoded by any ofSEQ ID NO: 1, 5, 10, and 12. In particular embodiments, the RNApolynucleotide is encoded by any of SEQ ID NO: 2, 4, 6 and 11. Inparticular embodiments, the RNA polynucleotide is encoded by any of SEQID NO: 7-9. In a particular embodiment, the RNA polynucleotide isencoded by SEQ ID NO: 3. In a particular embodiment, the RNApolynucleotide is encoded by SEQ ID NO: 13.

Nucleic acids (also referred to as polynucleotides) may be or mayinclude, for example, ribonucleic acids (RNAs), deoxyribonucleic acids(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNAhaving a β-D-ribo configuration, α-LNA having an α-L-ribo configuration(a diastereomer of LNA), 2′-amino-LNA having a 2′-aminofunctionalization, and 2′-amino-α-LNA having a 2′-aminofunctionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleicacids (CeNA) or chimeras or combinations thereof.

In some embodiments, polynucleotides of the present disclosure is orfunctions as a messenger RNA (mRNA). As used herein the term “messengerRNA” (mRNA) refers to any polynucleotide that encodes at least onepolypeptide (a naturally-occurring, non-naturally-occurring, or modifiedpolymer of amino acids) and can be translated to produce the encodedpolypeptide in vitro, in vivo, in situ or ex vivo.

Traditionally, the basic components of an mRNA molecule include at leastone coding region, a 5′ untranslated region (UTR), and a 3′ UTR. In someembodiments, the mRNA molecules further includes a 5′ cap. In someembodiments, the mRNA further includes a polyA tail. Polynucleotides ofthe present disclosure may function as mRNA but are distinguished fromwild-type mRNA in their functional and/or structural design featureswhich serve to overcome existing problems of effective polypeptideproduction using nucleic-acid based therapeutics. Antigenic polypeptides(antigens) of the present disclosure may be encoded by polynucleotidestranslated in vitro, referred to as “in vitro translated” (IVT)polynucleotides.

The RNA polynucleotides of the present disclosure may be or comprisevariant or mutant sequence. The term “polynucleotide variant” refers toa nucleotide molecule which differs in its nucleotide sequence from anative, wildtype, or reference sequence. The nucleotide sequencevariants may possess substitutions, deletions, and/or insertions atcertain positions within the nucleotide sequence, as compared to thecorresponding native, wildtype or reference sequence. In someembodiments, the nucleotide variants possess at least 80% identity(homology) to a native, wildtype or reference sequence, for example, atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (homology) to a native,wildtype or reference sequence.

In some embodiments, the RNA polynucleotide is encoded by a nucleic acidsequence having at least 80%-85% sequence identity to any of SEQ ID NO:1-14 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or199-212 (DENV), or 48-66 (ZIKV). In some embodiments, the RNApolynucleotide is encoded by a nucleic acid sequence having at least86%-90% sequence identity to any of SEQ ID NO: 1-13 (CHIKV), 16, 18, 20,22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or 199-212 (DENV), or 48-66(ZIKV). In some embodiments, the RNA polynucleotide is encoded by anucleic acid sequence having at least 91%-95% sequence identity to anyof SEQ ID NO: 1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33,34, 144-152 or 199-212 (DENV), or 48-66 (ZIKV). In some embodiments, theRNA polynucleotide is encoded by a nucleic acid sequence having at least96%-98% sequence identity to any of SEQ ID NO: 1-13 (CHIKV), 16, 18, 20,22, 24, 25, 27, 28, 30, 31, 33, 34, 144-152 or 199-212 (DENV), or 48-66(ZIKV). In some embodiments, the RNA polynucleotide is encoded by anucleic acid sequence having at least 99% sequence identity to any ofSEQ ID NO: 1-13 (CHIKV), 16, 18, 20, 22, 24, 25, 27, 28, 30, 31, 33, 34,144-152 or 199-212 (DENV), or 48-66 (ZIKV).

In some embodiments, a polynucleotide of the present disclosure, e.g.,polynucleotide variants, have less than 80% identity (homology) to anative, wildtype or reference sequence, for example, less than 80%, 79%,78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%,64%, 63%, 62%, 61%, 60% or less identity (homology) to a native,wildtype or reference sequence. In some embodiments, polynucleotide ofthe invention, e.g., polynucleotide variants, have about 65% to about85% identity to a native, wildtype or reference sequence, e.g., 65%-82%,67%-81%, or 66%-80% identity to a native, wildtype or referencesequence.

Polynucleotides of the present disclosure, in some embodiments, arecodon optimized. Codon optimization methods are known in the art and maybe used as provided herein. Codon optimization, in some embodiments, maybe used to match codon frequencies in target and host organisms toensure proper folding; bias GC content to increase mRNA stability orreduce secondary structures; minimize tandem repeat codons or base runsthat may impair gene construction or expression; customizetranscriptional and translational control regions; insert or removeprotein trafficking sequences; remove/add post translation modificationsites in encoded protein (e.g. glycosylation sites); add, remove orshuffle protein domains; insert or delete restriction sites; modifyribosome binding sites and mRNA degradation sites; adjust translationalrates to allow the various domains of the protein to fold properly; orto reduce or eliminate problem secondary structures within thepolynucleotide. Codon optimization tools, algorithms and services areknown in the art. Non-limiting examples include services from GeneArt(Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietarymethods. In some embodiments, the open reading frame (ORF) sequence isoptimized using optimization algorithms.

In some embodiments, a codon optimized sequence shares less than 95%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide. Insome embodiments, a codon optimized sequence shares less than 90%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide. Insome embodiments, a codon optimized sequence shares less than 85%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide. Insome embodiments, a codon optimized sequence shares less than 80%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide. Insome embodiments, a codon optimized sequence shares less than 75%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide.

In some embodiments, a codon optimized sequence shares between 65% and85% (e.g., between about 67% and about 85% or between about 67% andabout 80%) sequence identity to a naturally-occurring or wild-typesequence (e.g., a naturally-occurring or wild-type mRNA sequenceencoding a polypeptide or protein of interest (e.g., an antigenicprotein or polypeptide. In some embodiments, a codon optimized sequenceshares between 65% and 75 or about 80% sequence identity to anaturally-occurring or wild-type sequence (e.g., a naturally-occurringor wild-type mRNA sequence encoding a polypeptide or protein of interest(e.g., an antigenic protein or polypeptide.

In some embodiments, the RNA polynucleotides of the present disclosuremay further comprise sequence comprising or encoding additionalsequence, for example, one or more functional domain(s), one or morefurther regulatory sequence(s), an engineered 5′ cap.

Thus, in some embodiments, the RNA vaccines comprise a 5′UTR element, anoptionally codon optimized open reading frame, and a 3′UTR element, apoly(A) sequence and/or a polyadenylation signal wherein the RNA is notchemically modified.

In Vitro Transcription of RNA (e.g., mRNA)

The combination vaccine of the present disclosure comprise at least oneRNA polynucleotide, such as a mRNA (e.g., modified mRNA). mRNA, forexample, is transcribed in vitro from template DNA, referred to as an“in vitro transcription template.” In some embodiments, an in vitrotranscription template encodes a 5′ untranslated (UTR) region, containsan open reading frame, and encodes a 3′ UTR and a polyA tail. Theparticular nucleotide sequence composition and length of an in vitrotranscription template will depend on the mRNA encoded by the template.

A “5′ untranslated region” (UTR) refers to a region of an mRNA that isdirectly upstream (i.e., 5′) from the start codon (i.e., the first codonof an mRNA transcript translated by a ribosome) that does not encode apolypeptide.

A “3′ untranslated region” (UTR) refers to a region of an mRNA that isdirectly downstream (i.e., 3′) from the stop codon (i.e., the codon ofan mRNA transcript that signals a termination of translation) that doesnot encode a polypeptide.

An “open reading frame” is a continuous stretch of codons beginning witha start codon (e.g., methionine (ATG)), and ending with a stop codon(e.g., TAA, TAG or TGA) that encodes a polypeptide.

A “polyA tail” is a region of mRNA that is downstream, e.g., directlydownstream (i.e., 3′), from the 3′ UTR that contains multiple,consecutive adenosine monophosphates. A polyA tail may contain 10 to 300adenosine monophosphates. For example, a polyA tail may contain 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosinemonophosphates. In some embodiments, a polyA tail contains 50 to 250adenosine monophosphates. In a relevant biological setting (e.g., incells, in vivo) the poly(A) tail functions to protect mRNA fromenzymatic degradation, e.g., in the cytoplasm, and aids in transcriptiontermination, export of the mRNA from the nucleus and translation.

In some embodiments a codon optimized RNA may, for instance, be one inwhich the levels of G/C are enhanced. The G/C-content of nucleic acidmolecules may influence the stability of the RNA. RNA having anincreased amount of guanine (G) and/or cytosine (C) residues may befunctionally more stable than nucleic acids containing a large amount ofadenine (A) and thymine (T) or uracil (U) nucleotides. WO02/098443discloses a pharmaceutical composition containing an mRNA stabilized bysequence modifications in the translated region. Due to the degeneracyof the genetic code, the modifications work by substituting existingcodons for those that promote greater RNA stability without changing theresulting amino acid. The approach is limited to coding regions of theRNA.

Antigens/Antigenic Polypeptides

In some embodiments, the Chikungunya antigenic polypeptide is aChikungunya structural protein. The Chikungunya structural protein canbe a CHIKV envelope (E) protein or a CHIKV capsid (C) protein. In someembodiments, the Chikungunya structural protein can be a CHIKV E1, E2,E3, 6K, or capsid (C) protein. In one embodiment, the Chikungunyastructural protein is CHIKV E1. In another embodiment, the Chikungunyastructural protein is CHIKV E2. In another embodiment, the Chikungunyastructural protein is CHIKV E3. In another embodiment, the Chikungunyastructural protein is CHIKV C. In another embodiment, the Chikungunyastructural protein is CHIKV 6K.

In some embodiments, the Chikungunya antigenic polypeptide comprises thesequence of two or more Chikungunya structural proteins selected fromE1, E2, E3, 6K, and C. The antigenic polypeptide can comprise thesequence of any combination of CHIKV structural proteins, including, forexample, CHIKV E1 and E2; CHIKV E2 and E3; CHIKV E1 and E3; CHIKV E1,E2, and E3; CHIKV E1, E2, E3, and C; CHIKV E1, E2, E3, 6K, and C; CHIKVE1, 6K, E2; CHIKV E2, 6K, E3; CHIKV E1, 6K, E3; and CHIKV E1, E2, E3,and 6K proteins. In one particular embodiment, the Chikungunya antigenicpolypeptide comprises the sequence of the Chikungunya structuralpolyprotein: C-E3-E2-6K-E1.

In some embodiments, the Chikungunya antigenic polypeptide is a fragmentof a Chikungunya structural protein. The Chikungunya structural proteinfragment can be a CHIKV envelope (E) protein fragment or a CHIKV capsid(C) protein fragment. In some embodiments, the Chikungunya structuralprotein fragment can be a CHIKV E1, E2, E3, 6K, or capsid (C) proteinfragment. In one embodiment, the Chikungunya structural protein fragmentis CHIKV E1 fragment. In another embodiment, the Chikungunya structuralprotein fragment is CHIKV E2 fragment. In another embodiment, theChikungunya structural protein fragment is CHIKV E3 fragment. In anotherembodiment, the Chikungunya structural protein fragment is a CHIKV Cfragment. In another embodiment, the Chikungunya structural proteinfragment is a CHIKV 6K fragment.

In some embodiments, the Chikungunya antigenic polypeptide comprises thesequence of two or more Chikungunya structural protein fragmentsselected from E1, E2, E3, 6K, and C protein fragments. The antigenicpolypeptide can comprise the sequence of any combination of CHIKVstructural protein fragments, including, for example, CHIKV E1 and E2protein fragments; CHIKV E2 and E3 protein fragments; CHIKV E1 and E3protein fragments; CHIKV E1, E2, and E3 protein fragments; CHIKV E1, E2,E3, and C protein fragments; CHIKV E1, E2, E3, 6K, and C proteinfragments; CHIKV E1, 6K, and E2 protein fragments; CHIKV E2, 6K, and E3protein fragments; CHIKV E1, 6K, and E3 protein fragments; and CHIKV E1,E2, E3, and 6K protein fragments. In one particular embodiment, theChikungunya antigenic polypeptide comprises the sequence of a fragmentof the Chikungunya structural polyprotein: C-E3-E2-6K-E1.

In some embodiments, the Chikungunya antigenic polypeptide comprises thesequence of two or more Chikungunya structural proteins in which theproteins are a combination of full-length protein(s) and fragment(s)selected from E1, E2, E3, 6K, and C full-length protein(s) andfragment(s). The Chikungunya antigenic polypeptide may comprise thesequence of any combination of full-length protein(s) and fragment(s)including, for example, CHIKV E1 and E2 full-length protein(s) andfragment(s); CHIKV E2 and E3 full-length protein(s) and fragment(s);CHIKV E1 and E3 full-length protein(s) and fragment(s); CHIKV E1, E2,and E3 full-length protein(s) and fragment(s); CHIKV E1, E2, E3, and Cfull-length protein(s) and fragments; CHIKV E1, E2, E3, and 6Kfull-length protein(s) and fragment(s); CHIKV E1, E2, E3, 6K, and Cfull-length protein(s) and fragment(s); CHIKV E1, 6K, and E2 full-lengthprotein(s) and fragment(s); CHIKV E2, 6K, and E3 full-length protein(s)and fragment(s); and CHIKV E1, 6K, and E3 full-length protein(s) andfragment(s). In one particular embodiment, the Chikungunya antigenicpolypeptide comprises the sequence of the Chikungunya structuralpolyprotein: C-E3-E2-6K-E1 in which the proteins are a combination offull-length protein(s) and fragment(s).

The polypeptide antigens of the present disclosure can be one or morefull-length CHIKV protein antigens, one or more fragment antigens, oneor more epitope antigens or any combination of sequences thereof. Insome embodiments, the CHIKV antigenic polypeptide comprises 10-25 aminoacids. In some embodiments, the CHIKV antigenic polypeptide comprises26-50 amino acids. In some embodiments, the CHIKV antigenic polypeptidecomprises 51-100 amino acids. In some embodiments, the CHIKV antigenicpolypeptide comprises 101-200 amino acids. In some embodiments, theCHIKV antigenic polypeptide comprises 201-400 amino acids. In someembodiments, the CHIKV antigenic polypeptide comprises 401-500 aminoacids. In some embodiments, the CHIKV antigenic polypeptide comprises501-750 amino acids. In some embodiments, the CHIKV antigenicpolypeptide comprises 751-1000 amino acids. In some embodiments, theCHIKV antigenic polypeptide comprises 1001-1500 amino acids. In someembodiments, the CHIKV antigenic polypeptide comprises 1501-2000 aminoacids. In some embodiments, the CHIKV antigenic polypeptide comprises2001-4000 amino acids.

The polypeptide antigens of the present disclosure can be one or morefull-length DENV protein antigens, one or more fragment antigens, one ormore epitope antigens or any combination of sequences thereof. In someembodiments, the DENV antigenic polypeptide comprises 10-25 amino acids.In some embodiments, the DENV antigenic polypeptide comprises 26-50amino acids. In some embodiments, the DENV antigenic polypeptidecomprises 51-100 amino acids. In some embodiments, the DENV antigenicpolypeptide comprises 101-200 amino acids. In some embodiments, the DENVantigenic polypeptide comprises 201-400 amino acids. In someembodiments, the DENV antigenic polypeptide comprises 401-500 aminoacids. In some embodiments, the DENV antigenic polypeptide comprises501-750 amino acids. In some embodiments, the DENV antigenic polypeptidecomprises 751-1000 amino acids. In some embodiments, the DENV antigenicpolypeptide comprises 1001-1500 amino acids. In some embodiments, theDENV antigenic polypeptide comprises 1501-2000 amino acids. In someembodiments, the DENV antigenic polypeptide comprises 2001-4000 aminoacids.

The polypeptide antigens of the present disclosure can be one or morefull-length ZIKV protein antigens, one or more fragment antigens, one ormore epitope antigens or any combination of sequences thereof. In someembodiments, the ZIKV antigenic polypeptide comprises 10-25 amino acids.In some embodiments, the ZIKV antigenic polypeptide comprises 26-50amino acids. In some embodiments, the ZIKV antigenic polypeptidecomprises 51-100 amino acids. In some embodiments, the ZIKV antigenicpolypeptide comprises 101-200 amino acids. In some embodiments, the ZIKVantigenic polypeptide comprises 201-400 amino acids. In someembodiments, the ZIKV antigenic polypeptide comprises 401-500 aminoacids. In some embodiments, the ZIKV antigenic polypeptide comprises501-750 amino acids. In some embodiments, the ZIKV antigenic polypeptidecomprises 751-1000 amino acids. In some embodiments, the ZIKV antigenicpolypeptide comprises 1001-1500 amino acids. In some embodiments, theZIKV antigenic polypeptide comprises 1501-2000 amino acids. In someembodiments, the ZIKV antigenic polypeptide comprises 2001-4000 aminoacids.

The antigenic polypeptides include gene products, naturally occurringpolypeptides, synthetic or engineered polypeptides, mutant polypeptides,homologs, orthologs, paralogs, fragments and other equivalents,variants, and analogs of the foregoing. A polypeptide may be a singlemolecule or may be a multi-molecular complex such as a dimer, trimer ortetramer. Polypeptides may also comprise single chain or multichainpolypeptides such as antibodies or insulin and may be associated orlinked. Most commonly, disulfide linkages are found in multichainpolypeptides. The term polypeptide may also apply to amino acid polymersin which at least one amino acid residue is an artificial chemicalanalogue of a corresponding naturally-occurring amino acid.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native, wildtype, or reference sequence. Theamino acid sequence variants may possess substitutions, deletions,and/or insertions at certain positions within the amino acid sequence,as compared to a native, wildtype, or reference sequence. Ordinarily,variants possess at least 50% identity (homology) to a native, wildtype,or reference sequence. In some embodiments, variants possess at least80%, or at least 90% identical (homologous) to a native, wildtype, orreference sequence.

Examples of natural variants that are encompassed by the presentdisclosure include CHIKV, DENV, and ZIKV structural polypeptides fromdifferent CHIKV genotypes, lineages, strains, and isolates. Aphylogenetic tree of Chikungunya virus strains derived from completeconcatenated open reading frames for the nonstructural and structuralpolyproteins shows key envelope glycoprotein E1 amino acid substitutionsthat facilitated (Indian Ocean lineage) or prevented (Asian lineage)adaptation to Aedes albopictus. There are membrane-bound and secretedforms of E1 and E2, as well as the full length polyprotein antigen,which retains the protein's native conformation. Additionally, thedifferent Chikungunya genotypes can also yield different antigens, whichare functional in the constructs of the invention. There are severalChikungunya genotypes: Indian Ocean, East/Central/South African (ECSA),Asian, West African, and the Brazilian isolates (ECSA/Asian). Thus, forexample, natural variants that are encompassed by the present disclosureinclude, but is not limited to, CHIKV structural polypeptides from thefollowing strains and isolates: TA53, SA76, UG82, 37997, IND-06, Ross,S27, M-713424, E1-A226V, E1-T98, IND-63-WB1, Gibbs 63-263, TH35,1-634029, AF15561, IND-73-MH5, 653496, CO392-95, PO731460,MY0211MR/06/BP, SV0444-95, K0146-95, TSI-GSD-218-VR1, TSI-GSD-218, M127,M125, 6441-88, MY003IMR/06/BP, MY002IMR/06/BP, TR206/H804187,MY/06/37348, MY/06/37350, NC/2011-568, 1455-75, RSU1, 0706aTw,InDRE51CHIK, PR-S4, AMA2798/H804298, Hu/85/NR/001, PhH15483, 0706aTw,0802aTw, MY019IMR/06/BP, PR-S6, PER160/H803609, 99659, JKT23574,0811aTw, CHIK/SBY6/10, 2001908323-BDG E1, 2001907981-BDG E1,2004904899-BDG E1, 2004904879-BDG E1, 2003902452-BDG E1, DH130003,0804aTw, 2002918310-BDG E1, JC2012, chik-sy, 3807, 3462, Yap 13-2148,PR-S5, 0802aTw, MY019IMR/06/Bp, 0706aTw, PhH15483, Hu/85/NR/001,CHIKV-13-112A, InDRE4CHIK, 0806aTw, 0712aTw, 3412-78, Yap 13-2039,LEIV-CHIKV/Moscow/1, DH130003, and 20039.

In some embodiments “variant mimics” are provided. As used herein, theterm “variant mimic” is one which contains at least one amino acid thatwould mimic an activated sequence. For example, glutamate may serve as amimic for phosphoro-threonine and/or phosphoro-serine. Alternatively,variant mimics may result in deactivation or in an inactivated productcontaining the mimic, for example, phenylalanine may act as aninactivating substitution for tyrosine; or alanine may act as aninactivating substitution for serine.

“Homology” as it applies to amino acid sequences is defined as thepercentage of residues in the candidate amino acid sequence that areidentical with the residues in the amino acid sequence of a secondsequence after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent homology. Methods and computerprograms for the alignment are well known in the art. It is understoodthat homology depends on a calculation of percent identity but maydiffer in value due to gaps and penalties introduced in the calculation.By “homologs” as it applies to polypeptide sequences means thecorresponding sequence of other species having substantial identity to asecond sequence of a second species.

“Analogs” is meant to include polypeptide variants which differ by oneor more amino acid alterations, for example, substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

The present disclosure provides several types of compositions that arepolypeptide based, including variants and derivatives. These include,for example, substitutional, insertional, deletion and covalent variantsand derivatives. The term “derivative” is used synonymously with theterm “variant” but generally refers to a molecule that has been modifiedand/or changed in any way relative to a reference molecule or startingmolecule.

As such, polynucleotides encoding peptides or polypeptides containingsubstitutions, insertions and/or additions, deletions and covalentmodifications with respect to reference sequences, in particular thepolypeptide sequences disclosed herein, are included within the scope ofthis disclosure. “Substitutional variants” when referring topolypeptides are those that have at least one amino acid residue in anative or starting sequence removed and a different amino acid insertedin its place at the same position. Substitutions may be single, whereonly one amino acid in the molecule has been substituted, or they may bemultiple, where two or more amino acids have been substituted in thesame molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another, or the substitution of one acidicresidue such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Features” when referring to polypeptide or polynucleotide are definedas distinct amino acid sequence-based or nucleotide-based components ofa molecule respectively. Features of the polypeptides encoded by thepolynucleotides include surface manifestations, local conformationalshape, folds, loops, half-loops, domains, half-domains, sites, terminior any combination thereof.

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments is used synonymously with“amino acid residue” and “amino acid side chain.” As used herein whenreferring to polynucleotides the terms “site” as it pertains tonucleotide based embodiments is used synonymously with “nucleotide.” Asite represents a position within a peptide or polypeptide orpolynucleotide that may be modified, manipulated, altered, derivatizedor varied within the polypeptide or polynucleotide based molecules.

As used herein the terms “termini” or “terminus” when referring topolypeptides or polynucleotides refers to an extremity of a polypeptideor polynucleotide respectively. Such extremity is not limited only tothe first or final site of the polypeptide or polynucleotide but mayinclude additional amino acids or nucleotides in the terminal regions.Polypeptide-based molecules may be characterized as having both anN-terminus (terminated by an amino acid with a free amino group (NH2))and a C-terminus (terminated by an amino acid with a free carboxyl group(COOH)). Proteins are in some cases made up of multiple polypeptidechains brought together by disulfide bonds or by non-covalent forces(multimers, oligomers). These proteins have multiple N- and C-termini.Alternatively, the termini of the polypeptides may be modified such thatthey begin or end, as the case may be, with a non-polypeptide basedmoiety such as an organic conjugate.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest. For example, providedherein is any protein fragment (meaning a polypeptide sequence at leastone amino acid residue shorter than a reference polypeptide sequence butotherwise identical) of a reference protein 10, 20, 30, 40, 50, 60, 70,80, 90, 100 or greater than 100 amino acids in length. In anotherexample, any protein that includes a stretch of 20, 30, 40, 50, or 100amino acids which are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%identical to any of the sequences described herein can be utilized inaccordance with the disclosure. In some embodiments, a polypeptideincludes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in anyof the sequences provided or referenced herein.

Reference molecules (polypeptides or polynucleotides) may share acertain identity with the designed molecules (polypeptides orpolynucleotides). The term “identity” as known in the art, refers to arelationship between the sequences of two or more peptides, polypeptidesor polynucleotides, as determined by comparing the sequences. In theart, identity also means the degree of sequence relatedness between themas determined by the number of matches between strings of two or moreamino acid residues or nucleosides. Identity measures the percent ofidentical matches between the smaller of two or more sequences with gapalignments (if any) addressed by a particular mathematical model orcomputer program (e.g., “algorithms”). Identity of related peptides canbe readily calculated by known methods. Generally, variants of aparticular polynucleotide or polypeptide have at least 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% but less than 100% sequence identity to that particularreference polynucleotide or polypeptide as determined by sequencealignment programs and parameters described herein and known to thoseskilled in the art. Such tools for alignment include those of the BLASTsuite (Stephen F. Altschul, et al (1997), “Gapped BLAST and PSI-BLAST: anew generation of protein database search programs”, Nucleic Acids Res.25:3389-3402). A general global alignment technique based on dynamicprogramming is the Needleman-Wunsch algorithm. More recently a FastOptimal Global Sequence Alignment Algorithm (FOGSAA) has been developedthat purportedly produces global alignment of nucleotide and proteinsequences faster than other optimal global alignment methods, includingthe Needleman-Wunsch algorithm. Other tools are described herein,specifically in the definition of “identity” below.

As used herein, the term “homology” refers to the overall relatednessbetween polymeric molecules, e.g. between nucleic acid molecules (e.g.DNA molecules and/or RNA molecules) and/or between polypeptidemolecules. In some embodiments, polymeric molecules are considered to be“homologous” to one another if their sequences are at least 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%identical or similar. The term “homologous” necessarily refers to acomparison between at least two sequences (polynucleotide or polypeptidesequences). Two polynucleotide sequences are considered homologous ifthe polypeptides they encode are at least 50%, 60%, 70%, 80%, 90%, 95%,or even 99% for at least one stretch of at least 20 amino acids. In someembodiments, homologous polynucleotide sequences are characterized bythe ability to encode a stretch of at least 4-5 uniquely specified aminoacids. For polynucleotide sequences less than 60 nucleotides in length,homology is determined by the ability to encode a stretch of at least4-5 uniquely specified amino acids. Two protein sequences are consideredhomologous if the proteins are at least 50%, 60%, 70%, 80%, or 90%identical for at least one stretch of at least 20 amino acids.

The term “identity” refers to the overall relatedness between polymericmolecules, for example, between polynucleotide molecules (e.g. DNAmolecules and/or RNA molecules) and/or between polypeptide molecules.Calculation of the percent identity of two polynucleotide sequences, forexample, can be performed by aligning the two sequences for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second nucleic acid sequences for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Incertain embodiments, the length of a sequence aligned for comparisonpurposes is at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or 100% of thelength of the reference sequence. The nucleotides at correspondingnucleotide positions are then compared. When a position in the firstsequence is occupied by the same nucleotide as the correspondingposition in the second sequence, then the molecules are identical atthat position. The percent identity between the two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which needs to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

In some embodiments, the polypeptides further comprise additionalsequences or functional domains. For example, the CHIKV polypeptides ofthe present disclosure may comprise one or more linker sequences. Insome embodiments, the CHIKV of the present invention may comprise apolypeptide tag, such as an affinity tag (chitin binding protein (CBP),maltose binding protein (MBP), glutathione-S-transferase (GST), SBP-tag,Strep-tag, AviTag, Calmodulin-tag); solubilization tag; chromatographytag (polyanionic amino acid tag, such as FLAG-tag); epitope tag (shortpeptide sequences that bind to high-affinity antibodies, such as V5-tag,Myc-tag, VSV-tag, Xpress tag, E-tag, S-tag, and HA-tag); fluorescencetag (e.g., GFP). In some embodiments, the CHIKV of the present inventionmay comprise an amino acid tag, such as one or more lysines, histidines,or glutamates, which can be added to the polypeptide sequences (e.g., atthe N-terminal or C-terminal ends). Lysines can be used to increasepeptide solubility or to allow for biotinylation. Protein and amino acidtags are peptide sequences genetically grafted onto a recombinantprotein. Sequence tags are attached to proteins for various purposes,such as peptide purification, identification, or localization, for usein various applications including, for example, affinity purification,protein array, western blotting, immunofluorescence, andimmunoprecipitation. Such tags are subsequently removable by chemicalagents or by enzymatic means, such as by specific proteolysis or inteinsplicing.

Alternatively, amino acid residues located at the carboxy and aminoterminal regions of the amino acid sequence of a peptide or protein mayoptionally be deleted providing for truncated sequences. Certain aminoacids (e.g., C-terminal or N-terminal residues) may alternatively bedeleted depending on the use of the sequence, as for example, expressionof the sequence as part of a larger sequence which is soluble, or linkedto a solid support.

Multiprotein and Multicomponent Vaccines

The present disclosure encompasses CHIKV vaccines, DENV vaccines, ZIKVvaccines, CHIKV/DENV vaccines, CHIKV/ZIKV vaccines, ZIKV/DENV vaccines,and CHIKV/DENV/ZIKV vaccines comprising one or multiple RNA (e.g., mRNA)polynucleotides, each encoding a single antigenic polypeptide, as wellas vaccines comprising a single RNA polynucleotide encoding more thanone antigenic polypeptide (e.g., as a fusion polypeptide). Thus, itshould be understood that a vaccine composition comprising a RNApolynucleotide having an open reading frame encoding a first antigenicpolypeptide and a RNA polynucleotide having an open reading frameencoding a second antigenic polypeptide encompasses (a) vaccines thatcomprise a first RNA polynucleotide encoding a first antigenicpolypeptide and a second RNA polynucleotide encoding a second antigenicpolypeptide, and (b) vaccines that comprise a single RNA polynucleotideencoding a first and second antigenic polypeptide (e.g., as a fusionpolypeptide). RNA vaccines of the present disclosure, in someembodiments, comprise 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10), ormore, RNA polynucleotides having an open reading frame, each of whichencodes a different antigenic polypeptide (or a single RNApolynucleotide encoding 2-10, or more, different antigenicpolypeptides). In some embodiments, a RNA vaccine comprises a RNApolynucleotide having an open reading frame encoding a capsid protein, aRNA polynucleotide having an open reading frame encoding apremembrane/membrane protein, and a RNA polynucleotide having an openreading frame encoding a envelope protein. In some embodiments, a RNAvaccine comprises a RNA polynucleotide having an open reading frameencoding a capsid protein and a RNA polynucleotide having an openreading frame encoding a premembrane/membrane protein. In someembodiments, a RNA vaccine comprises a RNA polynucleotide having an openreading frame encoding a capsid protein and a RNA polynucleotide havingan open reading frame encoding a envelope protein. In some embodiments,a RNA vaccine comprises a RNA polynucleotide having an open readingframe encoding a premembrane/membrane protein and a RNA polynucleotidehaving an open reading frame encoding a envelope protein.

Signal Peptides

In some embodiments, a RNA polynucleotide encodes an antigenicpolypeptide fused to a signal peptide (e.g., SEQ ID NO: 125, 126, 128 or131). The signal peptide may be fused at the N-terminus or theC-terminus of the antigenic polypeptide. In some embodiments, antigenicpolypeptides encoded by CHIKV, DENV and/or ZIKV nucleic acids comprise asignal peptide. Signal peptides, comprising the N-terminal 15-60 aminoacids of proteins, are typically needed for the translocation across themembrane on the secretory pathway and thus universally control the entryof most proteins both in eukaryotes and prokaryotes to the secretorypathway. Signal peptides generally include of three regions: anN-terminal region of differing length, which usually comprisespositively charged amino acids; a hydrophobic region; and a shortcarboxy-terminal peptide region. In eukaryotes, the signal peptide of anascent precursor protein (pre-protein) directs the ribosome to therough endoplasmic reticulum (ER) membrane and initiates the transport ofthe growing peptide chain across it. The signal peptide is notresponsible for the final destination of the mature protein, however.Secretory proteins devoid of further address tags in their sequence areby default secreted to the external environment. Signal peptides arecleaved from precursor proteins by an endoplasmic reticulum(ER)-resident signal peptidase or they remain uncleaved and function asa membrane anchor. During recent years, a more advanced view of signalpeptides has evolved, showing that the functions and immunodominance ofcertain signal peptides are much more versatile than previouslyanticipated.

Proteins encoded by the ZIKV genome, e.g., the ZIKV Envelope protein,contain a signal peptide at the N-terminus to facilitate proteintargeting to the ER for processing. ER processing produces a matureEnvelope protein, wherein the signal peptide is cleaved, typically by asignal peptidase of the host cell. A signal peptide may also facilitatethe targeting of the protein to the cell membrane.

CHIKV vaccines, DENV vaccines, ZIKV vaccines, CHIKV/DENV vaccines,CHIKV/ZIKV vaccines, ZIKV/DENV vaccines, and CHIKV/DENV/ZIKV vaccines ofthe present disclosure may comprise, for example, RNA polynucleotidesencoding an artificial signal peptide, wherein the signal peptide codingsequence is operably linked to and is in frame with the coding sequenceof the CHIKV, DENV and/or ZIKV antigenic polypeptide. Thus, CHIKVvaccines, DENV vaccines, ZIKV vaccines, CHIKV/DENV vaccines, CHIKV/ZIKVvaccines, ZIKV/DENV vaccines, and CHIKV/DENV/ZIKV vaccines of thepresent disclosure, in some embodiments, produce an antigenicpolypeptide comprising a CHIKV, DENV and/or ZIKV antigenic polypeptidefused to a signal peptide. In some embodiments, a signal peptide isfused to the N-terminus of the CHIKV, DENV and/or ZIKV antigenicpolypeptide. In some embodiments, a signal peptide is fused to theC-terminus of the CHIKV, DENV and/or ZIKV antigenic polypeptide.

In some embodiments, the signal peptide fused to an antigenicpolypeptide is an artificial signal peptide. In some embodiments, anartificial signal peptide fused to an antigenic polypeptide encoded by aRNA vaccine is obtained from an immunoglobulin protein, e.g., an IgEsignal peptide or an IgG signal peptide. In some embodiments, a signalpeptide fused to an antigenic polypeptide encoded by a RNA vaccine is anIg heavy chain epsilon-1 signal peptide (IgE HC SP) having the sequenceof: MDWTWILFLVAAATRVHS (SEQ ID NO: 126). In some embodiments, a signalpeptide fused to a ZIKV antigenic polypeptide encoded by the ZIKV RNAvaccine is an IgG_(k) chain V-III region HAH signal peptide (IgG_(k) SP)having the sequence of METPAQLLFLLLLWLPDTTG (SEQ ID NO: 125). In someembodiments, a signal peptide fused to an antigenic polypeptide encodedby a RNA vaccine has an amino acid sequence set forth in SEQ ID NO: 125,126, 128 or 131. The examples disclosed herein are not meant to belimiting and any signal peptide that is known in the art to facilitatetargeting of a protein to ER for processing and/or targeting of aprotein to the cell membrane may be used in accordance with the presentdisclosure.

A signal peptide may have a length of 15-60 amino acids. For example, asignal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,or 60 amino acids. In some embodiments, a signal peptide may have alength of 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55,20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50,30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45,15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30,20-30, 25-30, 15-25, 20-25, or 15-20 amino acids.

Non-limiting examples of antigenic polypeptides fused to signalpeptides, which are encoded by a ZIKV RNA vaccine of the presentdisclosure, may be found in Table 31, SEQ ID NO: 48-59.

A signal peptide is typically cleaved from the nascent polypeptide atthe cleavage junction during ER processing, as illustrated in FIG. 26.The mature ZIKV antigenic polypeptide produce by a ZIKV RNA vaccine, forexample, typically does not comprise a signal peptide.

Chemical Modifications

In some embodiments, the RNA vaccines of the present disclosure, in someembodiments, comprise at least one ribonucleic acid (RNA) polynucleotidehaving an open reading frame encoding at least one CHIKV, DENV and/orZIKV antigenic polypeptide that comprises at least one chemicalmodification.

The terms “chemical modification” and “chemically modified” refer tomodification with respect to adenosine (A), guanosine (G), uridine (U),thymidine (T) or cytidine (C) ribonucleosides or deoxyribnucleosides inat least one of their position, pattern, percent or population.Generally, these terms do not refer to the ribonucleotide modificationsin naturally occurring 5′-terminal mRNA cap moieties. With respect to apolypeptide, the term “modification” refers to a modification relativeto the canonical set 20 amino acids. RNA polynucleotides, as providedherein, are also considered “modified” of they contain amino acidsubstitutions, insertions or a combination of substitutions andinsertions.

Polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides), in some embodiments, comprise various (more than one)different modifications. In some embodiments, a particular region of apolynucleotide contains one, two or more (optionally different)nucleoside or nucleotide modifications. In some embodiments, a modifiedRNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced toa cell or organism, exhibits reduced degradation in the cell ororganism, respectively, relative to an unmodified polynucleotide. Insome embodiments, a modified RNA polynucleotide (e.g., a modified mRNApolynucleotide), introduced into a cell or organism, may exhibit reducedimmunogenicity in the cell or organism, respectively (e.g., a reducedinnate response).

Modifications of polynucleotides include, without limitation, thosedescribed herein. Polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) may comprise modifications that arenaturally-occurring, non-naturally-occurring or the polynucleotide maycomprise a combination of naturally-occurring andnon-naturally-occurring modifications. Polynucleotides may include anyuseful modification, for example, of a sugar, a nucleobase, or aninternucleoside linkage (e.g., to a linking phosphate, to aphosphodiester linkage or to the phosphodiester backbone).

Polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides), in some embodiments, comprise non-natural modifiednucleotides that are introduced during synthesis or post-synthesis ofthe polynucleotides to achieve desired functions or properties. Themodifications may be present on an internucleotide linkages, purine orpyrimidine bases, or sugars. The modification may be introduced withchemical synthesis or with a polymerase enzyme at the terminal of achain or anywhere else in the chain. Any of the regions of apolynucleotide may be chemically modified.

The present disclosure provides for modified nucleosides and nucleotidesof a polynucleotide (e.g., RNA polynucleotides, such as mRNApolynucleotides). A “nucleoside” refers to a compound containing a sugarmolecule (e.g., a pentose or ribose) or a derivative thereof incombination with an organic base (e.g., a purine or pyrimidine) or aderivative thereof (also referred to herein as “nucleobase”). Anucleotide” refers to a nucleoside, including a phosphate group.Modified nucleotides may by synthesized by any useful method, such as,for example, chemically, enzymatically, or recombinantly, to include oneor more modified or non-natural nucleosides. Polynucleotides maycomprise a region or regions of linked nucleosides. Such regions mayhave variable backbone linkages. The linkages may be standardphosphodiester linkages, in which case the polynucleotides wouldcomprise regions of nucleotides.

Modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures. One example ofsuch non-standard base pairing is the base pairing between the modifiednucleotide inosine and adenine, cytosine or uracil. Any combination ofbase/sugar or linker may be incorporated into polynucleotides of thepresent disclosure.

The skilled artisan will appreciate that, except where otherwise noted,polynucleotide sequences set forth in the instant application willrecite “T”s in a representative DNA sequence but where the sequencerepresents RNA, the “T”s would be substituted for “U”s.

Modifications of polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) that are useful in the vaccines of the presentdisclosure include, but are not limited to the following:2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine;N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine; 1,2′-O-dimethyladenosine; 1-methyladenosine; 2′-O-methyladenosine;2′-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6isopentenyladenosine; 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine(phosphate); Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine;N6,2′-O-dimethyladenosine; N6,2′-O-dimethyladenosine;N6,N6,2′-O-trimethyladenosine; N6,N6-dimethyladenosine;N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine;N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine;2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;N1-methyl-adenosine; N6,N6 (dimethyl)adenine;N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine;2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine;2-(propyl)adenine; 2′-Amino-2′-deoxy-ATP; 2′-Azido-2′-deoxy-ATP;2′-Deoxy-2′-a-aminoadenosine TP; 2′-Deoxy-2′-a-azidoadenosine TP; 6(alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine;7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8(amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine;8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine;8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine;8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine;N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine;1-Deazaadenosine TP; 2′Fluoro-N6-Bz-deoxyadenosine TP;2′-OMe-2-Amino-ATP; 2′O-methyl-N6-Bz-deoxyadenosine TP;2′-a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP;2-Amino-ATP; 2′-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP;2′-b-Ethynyladenosine TP; 2-Bromoadenosine TP;2′-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP;2′-Deoxy-2′,2′-difluoroadenosine TP; 2′-Deoxy-2′-a-mercaptoadenosine TP;2′-Deoxy-2′-a-thiomethoxyadenosine TP; 2′-Deoxy-2′-b-aminoadenosine TP;2′-Deoxy-2′-b-azidoadenosine TP; 2′-Deoxy-2′-b-bromoadenosine TP;2′-Deoxy-2′-b-chloroadenosine TP; 2′-Deoxy-2′-b-fluoroadenosine TP;2′-Deoxy-2′-b-iodoadenosine TP; 2′-Deoxy-2′-b-mercaptoadenosine TP;2′-Deoxy-2′-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP;2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine;2-methylthio-adenine; 2-Trifluoromethyladenosine TP;3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP;3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP;3-Deazaadenosine TP; 4′-Azidoadenosine TP; 4′-Carbocyclic adenosine TP;4′-Ethynyladenosine TP; 5′-Homo-adenosine TP; 8-Aza-ATP;8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-DeazaadenosineTP; 2-aminopurine; 7-deaza-2,6-diaminopurine;7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine;2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;2-thiocytidine; 3-methylcytidine; 5-formylcytidine;5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;2′-O-methylcytidine; 2′-O-methylcytidine; 5,2′-O-dimethylcytidine;5-formyl-2′-O-methylcytidine; Lysidine; N4,2′-O-dimethylcytidine;N4-acetyl-2′-O-methylcytidine; N4-methylcytidine;N4,N4-Dimethyl-2′-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine;Pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine;2-(thio)cytosine; 2′-Amino-2′-deoxy-CTP; 2′-Azido-2′-deoxy-CTP;2′-Deoxy-2′-a-aminocytidine TP; 2′-Deoxy-2′-a-azidocytidine TP; 3(deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine;3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2′-O-dimethylcytidine;5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine;5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine;6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine;1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine;4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2′-anhydro-cytidine TPhydrochloride; 2′Fluor-N4-Bz-cytidine TP; 2′Fluoro-N4-Acetyl-cytidineTP; 2′-O-Methyl-N4-Acetyl-cytidine TP; 2′O-methyl-N4-Bz-cytidine TP;2′-a-Ethynylcytidine TP; 2′-a-Trifluoromethylcytidine TP;2′-b-Ethynylcytidine TP; 2′-b-Trifluoromethylcytidine TP;2′-Deoxy-2′,2′-difluorocytidine TP; 2′-Deoxy-2′-a-mercaptocytidine TP;2′-Deoxy-2′-a-thiomethoxycytidine TP; 2′-Deoxy-2′-b-aminocytidine TP;2′-Deoxy-2′-b-azidocytidine TP; 2′-Deoxy-2′-b-bromocytidine TP;2′-Deoxy-2′-b-chlorocytidine TP; 2′-Deoxy-2′-b-fluorocytidine TP;2′-Deoxy-2′-b-iodocytidine TP; 2′-Deoxy-2′-b-mercaptocytidine TP;2′-Deoxy-2′-b-thiomethoxycytidine TP; 2′-O-Methyl-5-(1-propynyl)cytidineTP; 3′-Ethynylcytidine TP; 4′-Azidocytidine TP; 4′-Carbocyclic cytidineTP; 4′-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP;5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidineTP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP;5-Ethynylcytidine TP; 5′-Homo-cytidine TP; 5-Methoxycytidine TP;5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidineTP; Pseudoisocytidine; 7-methylguanosine; N2,2′-O-dimethylguanosine;N2-methylguanosine; Wyosine; 1,2′-O-dimethylguanosine;1-methylguanosine; 2′-O-methylguanosine; 2′-O-ribosylguanosine(phosphate); 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate);7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine;Methylwyosine; N2,7-dimethylguanosine; N2,N2,2′-O-trimethylguanosine;N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine;N2,7,2′-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine;8-oxo-guanosine; N1-methyl-guanosine; α-thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2′-Amino-2′-deoxy-GTP;2′-Azido-2′-deoxy-GTP; 2′-Deoxy-2′-a-aminoguanosine TP;2′-Deoxy-2′-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine;6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7(deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine;7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8(halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine;8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; azaguanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine;1-methyl-6-thio-guanosine; 6-methoxy-guanosine;6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;N2-methyl-6-thio-guanosine; 1-Me-GTP; 2′Fluoro-N2-isobutyl-guanosine TP;2′O-methyl-N2-isobutyl-guanosine TP; 2′-a-Ethynylguanosine TP;2′-a-Trifluoromethylguanosine TP; 2′-b-Ethynylguanosine TP;2′-b-Trifluoromethylguanosine TP; 2′-Deoxy-2′,2′-difluoroguanosine TP;2′-Deoxy-2′-a-mercaptoguanosine TP; 2′-Deoxy-2′-a-thiomethoxyguanosineTP; 2′-Deoxy-2′-b-aminoguanosine TP; 2′-Deoxy-2′-b-azidoguanosine TP;2′-Deoxy-2′-b-bromoguanosine TP; 2′-Deoxy-2′-b-chloroguanosine TP;2′-Deoxy-2′-b-fluoroguanosine TP; 2′-Deoxy-2′-b-iodoguanosine TP;2′-Deoxy-2′-b-mercaptoguanosine TP; 2′-Deoxy-2′-b-thiomethoxyguanosineTP; 4′-Azidoguanosine TP; 4′-Carbocyclic guanosine TP;4′-Ethynylguanosine TP; 5′-Homo-guanosine TP; 8-bromo-guanosine TP;9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine;1,2′-O-dimethylinosine; 2′-O-methylinosine; 7-methylinosine;2′-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine;Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deazathymidine; deoxy-thymidine; 2′-O-methyluridine; 2-thiouridine;3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine;5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine;Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine;1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;1-methylpseduouridine; 1-methyl-pseudouridine; 2′-O-methyluridine;2′-O-methylpseudouridine; 2′-O-methyluridine; 2-thio-2′-O-methyluridine;3-(3-amino-3-carboxypropyl)uridine; 3,2′-O-dimethyluridine;3-Methyl-pseudo-Uridine TP; 4-thiouridine;5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methylester; 5,2′-O-dimethyluridine; 5,6-dihydro-uridine;5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2′-O-methyluridine;5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;5-carboxyhydroxymethyluridine methyl ester;5-carboxymethylaminomethyl-2′-O-methyluridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine;5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2′-O-methyluridine;5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine;5-methoxyuridine; 5-methyluridine, 5-methyl-2-thiouridine;5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine;5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyaceticacid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP;N1-methyl-pseudo-uridine; uridine 5-oxyacetic acid; uridine 5-oxyaceticacid methyl ester; 3-(3-Amino-3-carboxypropyl)-Uridine TP;5-(iso-Pentenylaminomethyl)-2-thiouridine TP;5-(iso-Pentenylaminomethyl)-2′-O-methyluridine TP;5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil;α-thio-uridine; 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1(aminocarbonylethylenyl)-2(thio)-pseudouracil; 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminocarbonylethylenyl)-pseudouracil; 1 substituted2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1substituted 4 (thio)pseudouracil; 1 substituted pseudouracil;1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil;1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP;1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP; 2(thio)pseudouracil; 2′ deoxy uridine; 2′ fluorouridine; 2-(thio)uracil;2,4-(dithio)psuedouracil; 2′ methyl, 2′amino, 2′azido,2′fluro-guanosine; 2′-Amino-2′-deoxy-UTP; 2′-Azido-2′-deoxy-UTP;2′-Azido-deoxyuridine TP; 2′-O-methylpseudouridine; 2′ deoxy uridine; 2′fluorouridine; 2′-Deoxy-2′-a-aminouridine TP; 2′-Deoxy-2′-a-azidouridineTP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil;5 (1,3-diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5(guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl)2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2(thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5(methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil;5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil;5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil;5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil;5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;5-(guanidiniumalkyl)uracil; 5-(halo)uracil;5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;5-(methoxycarbonylmethyl)-2-(thio)uracil;5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl)2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil;5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil;5-(methylaminomethyl)-2 (thio)uracil;5-(methylaminomethyl)-2,4(dithio)uracil;5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine;allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil;Pseudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP;1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine;1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine;1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine;2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine;2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;2-thio-dihydropseudouridine; 2-thio-dihydrouridine;2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine;(±)1-(2-Hydroxypropyl)pseudouridine TP;(2R)-1-(2-Hydroxypropyl)pseudouridine TP;(2S)-1-(2-Hydroxypropyl)pseudouridine TP;(E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP;(Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP;1-(2,2,2-Trifluoroethyl)-pseudo-UTP;1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP;1-(2,2-Diethoxyethyl)pseudouridine TP;1-(2,4,6-Trimethylbenzyl)pseudouridine TP;1-(2,4,6-Trimethyl-benzyl)pseudo-UTP;1-(2,4,6-Trimethyl-phenyl)pseudo-UTP;1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP;1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP;1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP;1-(3,4-Dimethoxybenzyl)pseudouridine TP;1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino-propyl)pseudo-UTP;1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP;1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP;1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP;1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP;1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP;1-(4-Iodobenzyl)pseudouridine TP;1-(4-Methanesulfonylbenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP;1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP;1-(4-Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP;1-(4-Nitro-benzyl)pseudo-UTP; 1(4-Nitro-phenyl)pseudo-UTP;1-(4-Thiomethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethylbenzyl)pseudouridine TP;1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP;1,6-Dimethyl-pseudo-UTP;1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridineTP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine TP;1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP;1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP;1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP;1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP;1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP;1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP;1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP;1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP;1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP;1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP;1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP;1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP;1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP;1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine TP;1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP;1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP;1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP;1-Methoxymethylpseudouridine TP;1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP;1-Methyl-6-(4-morpholino)-pseudo-UTP;1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substitutedphenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP;1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP;1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP;1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP;1-Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate-pseudo-UTP;1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP;1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP;1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP;1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP;1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP;1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP;1-Methyl-6-trifluoromethoxy-pseudo-UTP;1-Methyl-6-trifluoromethyl-pseudo-UTP; 1-MorpholinomethylpseudouridineTP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1-PivaloylpseudouridineTP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP;1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP;1-Thiomethoxymethylpseudouridine TP; 1-ThiomorpholinomethylpseudouridineTP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP;1-Vinylpseudouridine TP; 2,2′-anhydro-uridine TP; 2′-bromo-deoxyuridineTP; 2′-F-5-Methyl-2′-deoxy-UTP; 2′-OMe-5-Me-UTP; 2′-OMe-pseudo-UTP;2′-a-Ethynyluridine TP; 2′-a-Trifluoromethyluridine TP;2′-b-Ethynyluridine TP; 2′-b-Trifluoromethyluridine TP;2′-Deoxy-2′,2′-difluorouridine TP; 2′-Deoxy-2′-a-mercaptouridine TP;2′-Deoxy-2′-a-thiomethoxyuridine TP; 2′-Deoxy-2′-b-aminouridine TP;2′-Deoxy-2′-b-azidouridine TP; 2′-Deoxy-2′-b-bromouridine TP;2′-Deoxy-2′-b-chlorouridine TP; 2′-Deoxy-2′-b-fluorouridine TP;2′-Deoxy-2′-b-iodouridine TP; 2′-Deoxy-2′-b-mercaptouridine TP;2′-Deoxy-2′-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine;2-methoxyuridine; 2′-O-Methyl-5-(1-propynyl)uridine TP;3-Alkyl-pseudo-UTP; 4′-Azidouridine TP; 4′-Carbocyclic uridine TP;4′-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridineTP; 5-Cyanouridine TP; 5-Dimethylaminouridine TP; 5′-Homo-uridine TP;5-iodo-2′-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP;5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP;5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP;6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP;6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP;6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP;6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP;6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP;6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP;6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP;6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP;6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP;6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP;6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoicacid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; PseudouridineTP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid;Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid;Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid;Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid;Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid;Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine;undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine;1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;2,4,5-(trimethyl)phenyl; 2′ methyl, 2′amino, 2′azido, 2′fluro-cytidine;2′ methyl, 2′amino, 2′azido, 2′fluro-adenine; 2′methyl, 2′amino,2′azido, 2′fluro-uridine; 2′-amino-2′-deoxyribose;2-amino-6-Chloro-purine; 2-aza-inosinyl; 2′-azido-2′-deoxyribose;2′fluoro-2′-deoxyribose; 2′-fluoro-modified bases; 2′-O-methyl-ribose;2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl;3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl;5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine;6-(methyl)-7-(aza)indolyl; 6-chloro-purine;6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aza)indolyl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;Aminoindolyl; Anthracenyl;bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl;Isocarbostyrilyl; Isoguanisine; N2-substituted purines;N6-methyl-2-amino-purine; N6-substituted purines; N-alkylatedderivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl;Nitroindazolyl; Nitropyrazolyl; Nubularine; 06-substituted purines;O-alkylated derivative;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP;para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl;Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl;pyridopyrimidin-3-yl; pyridopyrimidin-3-yl,2-oxo-7-aminopyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5′-TP;2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine;pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP;Formycin B TP; Pyrrolosine TP; 2′-OH-ara-adenosine TP;2′-OH-ara-cytidine TP; 2′-OH-ara-uridine TP; 2′-OH-ara-guanosine TP;5-(2-carbomethoxyvinyl)uridine TP; andN6-(19-Amino-pentaoxanonadecyl)adenosine TP.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) includes a combination of at least two (e.g., 2,3, 4 or more) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in the polynucleotide (e.g.,RNA polynucleotide, such as mRNA polynucleotide) are selected from thegroup consisting of pseudouridine (ψ), N1-methylpseudouridine (m1ψ),2-thiouridine, 4′-thiouridine, 5-methylcytosine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine5-methyluridine, and 2′-O-methyluridine. In some embodiments, the polynucleotide (e.g., RNApolynucleotide, such as mRNA polynucleotide) includes a combination ofat least two (e.g., 2, 3, 4 or more) of the aforementioned modifiednucleobases.

In some embodiments, modified nucleobases in the polynucleotide (e.g.,RNA polynucleotide, such as mRNA polynucleotide) are selected from thegroup consisting of 1-methyl-pseudouridine (m1ψ), 5-methoxy-uridine(mo5U), 5-methyl-cytidine (m5C), pseudouridine (ψ), α-thio-guanosine andα-thio-adenosine. In some embodiments, the polynucleotide includes acombination of at least two (e.g., 2, 3, 4 or more) of theaforementioned modified nucleobases.

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) comprises pseudouridine (ψ) and5-methyl-cytidine (m5C). In some embodiments, the polynucleotide (e.g.,RNA polynucleotide, such as mRNA polynucleotide) comprises1-methyl-pseudouridine (m1ψ). In some embodiments, the polynucleotide(e.g., RNA polynucleotide, such as mRNA polynucleotide) comprises1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) comprises 2-thiouridine (s2U). In some embodiments, thepolynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide)comprises 2-thiouridine and 5-methyl-cytidine (m5C). In someembodiments, the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) comprises methoxy-uridine (mo5U). In some embodiments,the polynucleotide (e.g., RNA polynucleotide, such as mRNApolynucleotide) comprises 5-methoxy-uridine (mo5U) and 5-methyl-cytidine(m5C). In some embodiments, the polynucleotide (e.g., RNApolynucleotide, such as mRNA polynucleotide) comprises 2′-O-methyluridine. In some embodiments, the polynucleotide (e.g., RNApolynucleotide, such as mRNA polynucleotide) comprises 2′-O-methyluridine and 5-methyl-cytidine (m5C). In some embodiments, thepolynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide)comprises N6-methyl-adenosine (m6A). In some embodiments, thepolynucleotide (e.g., RNA polynucleotide, such as mRNA polynucleotide)comprises N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

In some embodiments, the polynucleotide (e.g., RNA polynucleotide, suchas mRNA polynucleotide) is uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a polynucleotide can be uniformly modified with5-methyl-cytidine (m5C), meaning that all cytosine residues in the mRNAsequence are replaced with 5-methyl-cytidine (m5C). Similarly, apolynucleotide can be uniformly modified for any type of nucleosideresidue present in the sequence by replacement with a modified residuesuch as any of those set forth above.

In some embodiments, the modified nucleobase is a modified cytosine.Examples of nucleobases and nucleosides having a modified cytosineinclude N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C),5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine(hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C),2-thio-5-methyl-cytidine.

In some embodiments, a modified nucleobase is a modified uridine.Example nucleobases and nucleosides having a modified uridine include5-cyano uridine or 4′-thio uridine.

In some embodiments, a modified nucleobase is a modified adenine.Example nucleobases and nucleosides having a modified adenine include7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A),N6-methyl-adenosine (m6A), and 2,6-Diaminopurine.

In some embodiments, a modified nucleobase is a modified guanine.Example nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0),7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G),1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵s²U),5-aminomethyl-2-thio-uridine (nm⁵s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine,5-taurinomethyl-2-thio-uridine(τm⁵s²U),1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U, i.e.,having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),5-methyl-2-thio-uridine (m⁵s²U), 1-methyl-4-thio-pseudouridine(m¹s⁴ψ),4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m³ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D),dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m⁵D),2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formylcytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴ ₂Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms² m⁶A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A)2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m¹Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o₂yW), hydroxywybutosine (OhyW), undermodifiedhydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m¹G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m^(2,7)G),N2,N2,7-dimethyl-guanosine (m^(2,2,7)G) 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m¹Im), 2′-O-ribosylguanosine (phosphate)(Gr(p)), 1-thio-guanosine, 06-methyl-guanosine, 2′-F-ara-guanosine, and2′-F-guanosine.

Methods of Treatment

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention and/or treatment of CHIKV,DENV, ZIKV, CHIKV/DENV (the combination of CHIKV and DENV, CHIKV/ZIKV(the combination of CHIKV and ZIKV), ZIKV and DENV (the combination ofZIKV and DENV), and CHIKV/DENV/ZIKV (the combination of CHIKV, DENV andZIKV) in humans and other mammals. CHIKV RNA (e.g. mRNA) vaccines, DENVRNA (e.g. mRNA) vaccines, ZIKV RNA (e.g. mRNA) vaccines, CHIKV/DENV RNA(e.g. mRNA) vaccines, CHIKV/ZIKV RNA (e.g. mRNA) vaccines, ZIKV/DENV RNA(e.g. mRNA) vaccines, and CHIKV/DENV/ZIKV RNA (e.g. mRNA) vaccines canbe used as therapeutic or prophylactic agents. They may be used inmedicine to prevent and/or treat infectious disease. In exemplaryaspects, the vaccines, of the present disclosure are used to provideprophylactic protection from CHIKV, DENV, ZIKV or any combination of twoor three of the foregoing viruses. Prophylactic protection from CHIKV,DENV and/or ZIKV can be achieved following administration of a CHIKV,DENV and/or ZIKV vaccine or combination vaccine, of the presentdisclosure. Vaccines (including combination vaccines) can beadministered once, twice, three times, four times or more but it islikely sufficient to administer the vaccine once (optionally followed bya single booster). It is possible, although less desirable, toadminister the vaccine to an infected individual to achieve atherapeutic response. Dosing may need to be adjusted accordingly.

Broad Spectrum Vaccines

It is envisioned that there may be situations where persons are at riskfor infection with more than one strain of CHIKV, DENV and/or ZIKV(e.g., more than one strain of CHIKV, more than one strain of DENV,and/or more than one strain of ZIKV). RNA (e.g., mRNA) therapeuticvaccines are particularly amenable to combination vaccination approachesdue to a number of factors including, but not limited to, speed ofmanufacture, ability to rapidly tailor vaccines to accommodate perceivedgeographical threat, and the like. Moreover, because the vaccinesutilize the human body to produce the antigenic protein, the vaccinesare amenable to the production of larger, more complex antigenicproteins, allowing for proper folding, surface expression, antigenpresentation, etc. in the human subject. To protect against more thanone strain of CHIKV, DENV and/or ZIKV, a vaccine (including acombination vaccine) can be administered that includes RNA encoding atleast one antigenic polypeptide protein (or antigenic portion thereof)of a first CHIKV, DENV and/or ZIKV and further includes RNA encoding atleast one antigenic polypeptide protein (or antigenic portion thereof)of a second CHIKV, DENV and/or ZIKV. RNAs (mRNAs) can be coformulated,for example, in a single lipid nanoparticle (LNP) or can be formulatedin separate LNPs destined for co-administration.

A method of eliciting an immune response in a subject against a CHIKV,DENV and/or ZIKV is provided in aspects of the invention. The methodinvolves administering to the subject a CHIKV, DENV and/or ZIKV RNAvaccine comprising at least one RNA polynucleotide having an openreading frame encoding at least one CHIKV, DENV and/or ZIKV antigenicpolypeptide or an immunogenic fragment thereof, thereby inducing in thesubject an immune response specific to CHIKV, DENV and/or ZIKV antigenicpolypeptide or an immunogenic fragment thereof, wherein anti-antigenicpolypeptide antibody titer in the subject is increased followingvaccination relative to anti-antigenic polypeptide antibody titer in asubject vaccinated with a prophylactically effective dose of atraditional vaccine against the CHIKV, DENV and/or ZIKV. An“anti-antigenic polypeptide antibody” is a serum antibody the bindsspecifically to the antigenic polypeptide.

A prophylactically effective dose is a therapeutically effective dosethat prevents infection with the virus at a clinically acceptable level.In some embodiments the therapeutically effective dose is a dose listedin a package insert for the vaccine. A traditional vaccine, as usedherein, refers to a vaccine other than the mRNA vaccines of theinvention. For instance, a traditional vaccine includes but is notlimited to live microorganism vaccines, killed microorganism vaccines,subunit vaccines, protein antigen vaccines, DNA vaccines, etc.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 1 log to 10 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theCHIKV, DENV and/or ZIKV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 1 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theCHIKV, DENV and/or ZIKV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 2 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theCHIKV, DENV and/or ZIKV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 3 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theCHIKV, DENV and/or ZIKV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 5 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theCHIKV, DENV and/or ZIKV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 10 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theCHIKV, DENV and/or ZIKV.

A method of eliciting an immune response in a subject against a CHIKV,DENV and/or ZIKV is provided in other aspects of the invention. Themethod involves administering to the subject a CHIKV, DENV and/or ZIKVRNA vaccine comprising at least one RNA polynucleotide having an openreading frame encoding at least one CHIKV, DENV and/or ZIKV antigenicpolypeptide or an immunogenic fragment thereof, thereby inducing in thesubject an immune response specific to CHIKV, DENV and/or ZIKV antigenicpolypeptide or an immunogenic fragment thereof, wherein the immuneresponse in the subject is equivalent to an immune response in a subjectvaccinated with a traditional vaccine against the CHIKV, DENV and/orZIKV at 2 times to 100 times the dosage level relative to the RNAvaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine attwice the dosage level relative to the CHIKV, DENV and/or ZIKV RNAvaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine atthree times the dosage level relative to the CHIKV, DENV and/or ZIKV RNAvaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at4 times the dosage level relative to the CHIKV, DENV and/or ZIKVvaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at5 times the dosage level relative to the CHIKV, DENV and/or ZIKV RNAvaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at10 times the dosage level relative to the CHIKV, DENV and/or ZIKV RNAvaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at50 times the dosage level relative to the CHIKV, DENV and/or ZIKV RNAvaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at100 times the dosage level relative to the CHIKV, DENV and/or ZIKV RNAvaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at10 times to 1000 times the dosage level relative to the CHIKV, DENVand/or ZIKV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at100 times to 1000 times the dosage level relative to the CHIKV, DENVand/or ZIKV RNA vaccine

In other embodiments the immune response is assessed by determining[protein] antibody titer in the subject.

In other aspects the present disclosure is a method of eliciting animmune response in a subject against a CHIKV, DENV and/or ZIKV byadministering to the subject a CHIKV, DENV and/or ZIKV RNA vaccinecomprising at least one RNA polynucleotide having an open reading frameencoding at least one CHIKV, DENV and/or ZIKV antigenic polypeptide oran immunogenic fragment thereof, thereby inducing in the subject animmune response specific to CHIKV, DENV and/or ZIKV antigenicpolypeptide or an immunogenic fragment thereof, wherein the immuneresponse in the subject is induced 2 days to 10 weeks earlier relativeto an immune response induced in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theCHIKV, DENV and/or ZIKV. In some embodiments the immune response in thesubject is induced in a subject vaccinated with a prophylacticallyeffective dose of a traditional vaccine at 2 times to 100 times thedosage level relative to the RNA vaccine.

In some embodiments the immune response in the subject is induced 2 daysearlier relative to an immune response induced in a subject vaccinatedwith a prophylactically effective dose of a traditional vaccine.

In some embodiments the immune response in the subject is induced 3 daysearlier relative to an immune response induced in a subject vaccinated aprophylactically effective dose of a traditional vaccine.

In some embodiments the immune response in the subject is induced 1 weekearlier relative to an immune response induced in a subject vaccinatedwith a prophylactically effective dose of a traditional vaccine.

In some embodiments the immune response in the subject is induced 2weeks earlier relative to an immune response induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine.

In some embodiments the immune response in the subject is induced 3weeks earlier relative to an immune response induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine.

In some embodiments the immune response in the subject is induced 5weeks earlier relative to an immune response induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine.

In some embodiments the immune response in the subject is induced 10weeks earlier relative to an immune response induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine.

Also provided herein are methods of eliciting an immune response in asubject against a CHIKV, DENV and/or ZIKV by administering to thesubject a CHIKV, DENV and/or ZIKV RNA vaccine having an open readingframe encoding a first antigenic polypeptide, wherein the RNApolynucleotide does not include a stabilization element, and wherein anadjuvant is not coformulated or co-administered with the vaccine.

Therapeutic and Prophylactic Compositions

Provided herein are compositions, methods, kits and reagents for theprevention, treatment or diagnosis of Chikungunya virus in humans andother mammals, for example. The active therapeutic agents of the presentdisclosure include the CHIKV, DENV and/or ZIKV RNA vaccines, includingcombination RNA vaccines), cells containing CHIKV, DENV and/or ZIKV RNAvaccines, including combination RNA vaccines), and antigenicpolypeptides translated from the polynucleotides comprising the RNAvaccines. CHIKV, DENV and/or ZIKV RNA vaccines, including combinationRNA vaccines) can be used as therapeutic or prophylactic agents. Theymay be used in medicine and/or for the priming of immune effector cells,for example, to activate peripheral blood mononuclear cells (PBMCs) exvivo, which are then infused (re-infused) into a subject.

In some embodiments, a vaccines, including a combination vaccine,containing RNA polynucleotides, e.g., mRNA, as described herein can beadministered to a subject (e.g., a mammalian subject, such as a humansubject), and the RNA polynucleotides are translated in vivo to producean antigenic polypeptide.

The CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNAvaccines, may be induced for translation of a polypeptide (e.g., antigenor immunogen) in a cell, tissue or organism. Such translation can be invivo, ex vivo, in culture or in vitro. The cell, tissue or organism iscontacted with an effective amount of a composition containing a CHIKV,DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, thatcontains a polynucleotide that has at least one a translatable regionencoding an antigenic polypeptide.

An “effective amount” of the CHIKV, DENV and/or ZIKV RNA vaccines,including combination RNA vaccines, is provided based, at least in part,on the target tissue, target cell type, means of administration,physical characteristics of the polynucleotide (e.g., size, and extentof modified nucleosides) and other components of the CHIKV, DENV and/orZIKV RNA vaccines, including combination RNA vaccines, and otherdeterminants. In general, CHIKV, DENV and/or ZIKV RNA vaccines,including combination RNA vaccines, provides an induced or boostedimmune response as a function of antigen production in the cell,preferably more efficient than a composition containing a correspondingunmodified polynucleotide encoding the same antigen or a peptideantigen. Increased antigen production may be demonstrated by increasedcell transfection (the percentage of cells transfected with the RNAvaccine), increased protein translation from the polynucleotide,decreased nucleic acid degradation (as demonstrated, for example, byincreased duration of protein translation from a modifiedpolynucleotide), or altered antigen specific immune response of the hostcell.

In some embodiments, RNA vaccines (including polynucleotides and theirencoded polypeptides) and cells comprising the RNA vaccines inaccordance with the present disclosure may be used for the treatment ofChikungunya virus, Dengue virus, Zika virus, or any combination of twoor three of the foregoing viruses.

CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNAvaccines, may be administered prophylactically or therapeutically aspart of an active immunization scheme to healthy individuals or early ininfection during the incubation phase or during active infection afteronset of symptoms. In some embodiments, the amount of RNA vaccine of thepresent disclosure provided to a cell, a tissue or a subject may be anamount effective for immune prophylaxis.

CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNAvaccines, may be administered with other prophylactic or therapeuticcompounds. As a non-limiting example, a prophylactic or therapeuticcompound may be an adjuvant or a booster. As used herein, when referringto a prophylactic composition, such as a vaccine, the term “booster”refers to an extra administration of the prophylactic (vaccine)composition. A booster (or booster vaccine) may be given after anearlier administration of the prophylactic composition. The time ofadministration between the initial administration of the prophylacticcomposition and the booster may be, but is not limited to, 1 minute, 2minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours,12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days,3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8months, 9 months, 10 months, 11 months, 1 year, 15 months, 18 months, 21months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99years, and any time period in-between.

In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, includingcombination RNA vaccines, may be administered intramuscularly orintradermally, similarly to the administration of inactivated vaccinesknown in the art.

The CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNAvaccines, may be utilized in various settings depending on theprevalence of the infection or the degree or level of unmet medicalneed. As a non-limiting example, the RNA vaccines may be utilized totreat and/or prevent infectious disease caused by Chikungunya virus. RNAvaccines have superior properties in that they produce much largerantibody titers and produce responses early than commercially availableanti-virals.

Provided herein are pharmaceutical compositions including CHIKV, DENVand/or ZIKV RNA vaccines, including combination RNA vaccines and RNAvaccine compositions and/or complexes optionally in combination with oneor more pharmaceutically acceptable excipients.

CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNAvaccines, may be formulated or administered alone or in conjunction withone or more other components. For instance, CHIKV, DENV and/or ZIKV RNAvaccines, including combination RNA vaccines (vaccine compositions) maycomprise other components including, but not limited to, adjuvants. Insome embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, includingcombination RNA vaccines, do not include an adjuvant (they are adjuvantfree).

CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNAvaccines, may be formulated or administered in combination with one ormore pharmaceutically-acceptable excipients. In some embodiments,vaccine compositions comprise at least one additional active substance,such as, for example, a therapeutically-active substance, aprophylactically-active substance, or a combination of both. Vaccinecompositions may be sterile, pyrogen-free or both sterile andpyrogen-free. General considerations in the formulation and/ormanufacture of pharmaceutical agents, such as vaccine compositions, maybe found, for example, in Remington: The Science and Practice ofPharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporatedherein by reference in its entirety).

In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, includingcombination RNA vaccines, are administered to humans, human patients orsubjects. For the purposes of the present disclosure, the phrase “activeingredient” generally refers to the RNA vaccines or the polynucleotidescontained therein, for example, RNA polynucleotides (e.g., mRNApolynucleotides) encoding CHIKV, DENV and/or ZIKV antigenicpolypeptides.

Formulations of the vaccine compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient (e.g., mRNA polynucleotide) intoassociation with an excipient and/or one or more other accessoryingredients, and then, if necessary and/or desirable, dividing, shapingand/or packaging the product into a desired single- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNAvaccines, can be formulated using one or more excipients to: (1)increase stability; (2) increase cell transfection; (3) permit thesustained or delayed release (e.g., from a depot formulation); (4) alterthe biodistribution (e.g., target to specific tissues or cell types);(5) increase the translation of encoded protein in vivo; and/or (6)alter the release profile of encoded protein (antigen) in vivo. Inaddition to traditional excipients such as any and all solvents,dispersion media, diluents, or other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, excipients can include, withoutlimitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with CHIKV, DENV and/or ZIKV RNA vaccines, includingcombination RNA vaccines, (e.g., for transplantation into a subject),hyaluronidase, nanoparticle mimics and combinations thereof.

Stabilizing Elements

Naturally-occurring eukaryotic mRNA molecules have been found to containstabilizing elements, including, but not limited to untranslated regions(UTR) at their 5′-end (5′UTR) and/or at their 3′-end (3′UTR), inaddition to other structural features, such as a 5′-cap structure or a3′-poly(A) tail. Both the 5′UTR and the 3′UTR are typically transcribedfrom the genomic DNA and are elements of the premature mRNA.Characteristic structural features of mature mRNA, such as the 5′-capand the 3′-poly(A) tail are usually added to the transcribed (premature)mRNA during mRNA processing. The 3′-poly(A) tail is typically a stretchof adenine nucleotides added to the 3′-end of the transcribed mRNA. Itcan comprise up to about 400 adenine nucleotides. In some embodimentsthe length of the 3′-poly(A) tail may be an essential element withrespect to the stability of the individual mRNA.

In some embodiments the RNA vaccine may include one or more stabilizingelements. Stabilizing elements may include for instance a histonestem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has beenidentified. It is associated with the histone stem-loop at the 3′-end ofthe histone messages in both the nucleus and the cytoplasm. Itsexpression level is regulated by the cell cycle; it is peaks during theS-phase, when histone mRNA levels are also elevated. The protein hasbeen shown to be essential for efficient 3′-end processing of histonepre-mRNA by the U7 snRNP. SLBP continues to be associated with thestem-loop after processing, and then stimulates the translation ofmature histone mRNAs into histone proteins in the cytoplasm. The RNAbinding domain of SLBP is conserved through metazoa and protozoa; itsbinding to the histone stem-loop depends on the structure of the loop.The minimum binding site includes at least three nucleotides 5′ and twonucleotides 3′ relative to the stem-loop.

In some embodiments, the RNA vaccines include a coding region, at leastone histone stem-loop, and optionally, a poly(A) sequence orpolyadenylation signal. The poly(A) sequence or polyadenylation signalgenerally should enhance the expression level of the encoded protein.The encoded protein, in some embodiments, is not a histone protein, areporter protein (e.g. Luciferase, GFP, EGFP, β-Galactosidase, EGFP), ora marker or selection protein (e.g. alpha-Globin, Galactokinase andXanthine:guanine phosphoribosyl transferase (GPT)).

In some embodiments, the combination of a poly(A) sequence orpolyadenylation signal and at least one histone stem-loop, even thoughboth represent alternative mechanisms in nature, acts synergistically toincrease the protein expression beyond the level observed with either ofthe individual elements. It has been found that the synergistic effectof the combination of poly(A) and at least one histone stem-loop doesnot depend on the order of the elements or the length of the poly(A)sequence.

In some embodiments, the RNA vaccine does not comprise a histonedownstream element (HDE). “Histone downstream element” (HDE) includes apurine-rich polynucleotide stretch of approximately 15 to 20 nucleotides3′ of naturally occurring stem-loops, representing the binding site forthe U7 snRNA, which is involved in processing of histone pre-mRNA intomature histone mRNA. Ideally, the inventive nucleic acid does notinclude an intron.

In some embodiments, the RNA vaccine may or may not contain a enhancerand/or promoter sequence, which may be modified or unmodified or whichmay be activated or inactivated. In some embodiments, the histonestem-loop is generally derived from histone genes, and includes anintramolecular base pairing of two neighbored partially or entirelyreverse complementary sequences separated by a spacer, consisting of ashort sequence, which forms the loop of the structure. The unpaired loopregion is typically unable to base pair with either of the stem loopelements. It occurs more often in RNA, as is a key component of many RNAsecondary structures, but may be present in single-stranded DNA as well.Stability of the stem-loop structure generally depends on the length,number of mismatches or bulges, and base composition of the pairedregion. In some embodiments, wobble base pairing (non-Watson-Crick basepairing) may result. In some embodiments, the at least one histonestem-loop sequence comprises a length of 15 to 45 nucleotides.

In other embodiments the RNA vaccine may have one or more AU-richsequences removed. These sequences, sometimes referred to as AURES aredestabilizing sequences found in the 3′UTR. The AURES may be removedfrom the RNA vaccines. Alternatively the AURES may remain in the RNAvaccine.

Nanoparticle Formulations

In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, includingcombination RNA vaccines, are formulated in a nanoparticle. In someembodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combinationRNA vaccines, are formulated in a lipid nanoparticle. In someembodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combinationRNA vaccines, are formulated in a lipid-polycation complex, referred toas a cationic lipid nanoparticle. The formation of the lipidnanoparticle may be accomplished by methods known in the art and/or asdescribed in U.S. Pub. No. 20120178702, herein incorporated by referencein its entirety. As a non-limiting example, the cationic lipidnanoparticle may include a cationic peptide or a polypeptide such as,but not limited to, polylysine, polyornithine and/or polyarginine andthe cationic peptides described in International Pub. No. WO2012013326or US Patent Pub. No. US20130142818; each of which is hereinincorporated by reference in its entirety. In some embodiments, CHIKV,DENV and/or ZIKV RNA vaccines, including combination RNA vaccines, areformulated in a lipid nanoparticle that includes a non-cationic lipidsuch as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

A lipid nanoparticle formulation may be influenced by, but not limitedto, the selection of the cationic lipid component, the degree ofcationic lipid saturation, the nature of the PEGylation, ratio of allcomponents and biophysical parameters such as size. For example, thelipid nanoparticle formulation may be composed of 57.1% cationic lipid,7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4%PEG-c-DMA. (Semple et al., Nature Biotech. 2010 28:172-176; hereinincorporated by reference in its entirety). Altering the composition ofthe cationic lipid can more effectively deliver RNA to various antigenpresenting cells (Basha et al. Mol Ther. 2011 19:2186-2200; hereinincorporated by reference in its entirety).

In some embodiments, lipid nanoparticle formulations may comprise 35 to45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipidand/or 55% to 65% cationic lipid. In some embodiments, the ratio oflipid to RNA (e.g., mRNA) in lipid nanoparticles may be 5:1 to 20:1,10:1 to 25:1, 15:1 to 30:1 and/or at least 30:1.

In some embodiments, the ratio of PEG in the lipid nanoparticleformulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the lipid nanoparticleformulations. As a non-limiting example, lipid nanoparticle formulationsmay contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5%to 5.0% and/or 3.0% to 6.0% of the lipid molar ratio of PEG-c-DOMG(R-3-[(ω-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine)(also referred to herein as PEG-DOMG) as compared to the cationic lipid,DSPC and cholesterol. In some embodiments, the PEG-c-DOMG may bereplaced with a PEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG(1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In some embodiments, the CHIKV, DENV and/or ZIKV RNA vaccines, includingcombination RNA vaccine formulation is a nanoparticle that comprises atleast one lipid. The lipid may be selected from, but is not limited to,DLin-DMA, Dlin-K-DMA, 98N12-5, C12-200, Dlin-MC3-DMA, Dlin-KC2-DMA,DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. Insome embodiments, the lipid may be a cationic lipid such as, but notlimited to, Dlin-DMA, Dlin-D-DMA, Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA andamino alcohol lipids. The amino alcohol cationic lipid may be the lipidsdescribed in and/or made by the methods described in US PatentPublication No. US20130150625, herein incorporated by reference in itsentirety. As a non-limiting example, the cationic lipid may be2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US20130150625); or any pharmaceutically acceptable saltor stereoisomer thereof.

Lipid nanoparticle formulations typically comprise a lipid, inparticular, an ionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In some embodiments, a lipid nanoparticle formulation consistsessentially of (i) at least one lipid selected from the group consistingof 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) aneutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) asterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG orPEG-cDMA, in a molar ratio of 20-60% cationic lipid: 5-25% neutrallipid: 25-55% sterol; 0.5-15% PEG-lipid.

In some embodiments, a lipid nanoparticle formulation includes 25% to75% on a molar basis of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., 35 to65%, 45 to 65%, 60%, 57.5%, 50% or 40% on a molar basis.

In some embodiments, a lipid nanoparticle formulation includes 0.5% to15% on a molar basis of the neutral lipid, e.g., 3 to 12%, 5 to 10% or15%, 10%, or 7.5% on a molar basis. Examples of neutral lipids include,without limitation, DSPC, POPC, DPPC, DOPE and SM. In some embodiments,the formulation includes 5% to 50% on a molar basis of the sterol (e.g.,15 to 45%, 20 to 40%, 40%, 38.5%, 35%, or 31% on a molar basis. Anon-limiting example of a sterol is cholesterol. In some embodiments, alipid nanoparticle formulation includes 0.5% to 20% on a molar basis ofthe PEG or PEG-modified lipid (e.g., 0.5 to 10%, 0.5 to 5%, 1.5%, 0.5%,1.5%, 3.5%, or 5% on a molar basis. In some embodiments, a PEG or PEGmodified lipid comprises a PEG molecule of an average molecular weightof 2,000 Da. In some embodiments, a PEG or PEG modified lipid comprisesa PEG molecule of an average molecular weight of less than 2,000, forexample around 1,500 Da, around 1,000 Da, or around 500 Da. Non-limitingexamples of PEG-modified lipids include PEG-distearoyl glycerol(PEG-DMG) (also referred herein as PEG-C14 or C14-PEG), PEG-cDMA(further discussed in Reyes et al. J. Controlled Release, 107, 276-287(2005) the contents of which are herein incorporated by reference in itsentirety).

In some embodiments, lipid nanoparticle formulations include 25-75% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (Dlin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% ofthe neutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 35-65% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (Dlin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of theneutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 45-65% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (Dlin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of theneutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 60% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (Dlin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.5% of theneutral lipid, 31% of the sterol, and 1.5% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 50% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (Dlin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of theneutral lipid, 38.5% of the sterol, and 1.5% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 50% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (Dlin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 10% of theneutral lipid, 35% of the sterol, 4.5% or 5% of the PEG or PEG-modifiedlipid, and 0.5% of the targeting lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 40% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (Dlin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 15% of theneutral lipid, 40% of the sterol, and 5% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 57.2% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (Dlin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (Dlin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 7.1% of theneutral lipid, 34.3% of the sterol, and 1.4% of the PEG or PEG-modifiedlipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 57.5% of acationic lipid selected from the PEG lipid is PEG-cDMA (PEG-cDMA isfurther discussed in Reyes et al. (J. Controlled Release, 107, 276-287(2005), the contents of which are herein incorporated by reference inits entirety), 7.5% of the neutral lipid, 31.5% of the sterol, and 3.5%of the PEG or PEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations consistsessentially of a lipid mixture in molar ratios of 20-70% cationic lipid:5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid. Insome embodiments, lipid nanoparticle formulations consists essentiallyof a lipid mixture in a molar ratio of 20-60% cationic lipid: 5-25%neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.

In some embodiments, the molar lipid ratio is 50/10/38.5/1.5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG, PEG-DSG or PEG-DPG), 57.2/7.1134.3/1.4 (mol % cationiclipid/neutral lipid, e.g., DPPC/Chol/PEG-modified lipid, e.g.,PEG-cDMA), 40/15/40/5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 50/10/35/4.5/0.5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DSG), 50/10/35/5 (cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 40/10/40/10 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG or PEG-cDMA), 35/15/40/10 (mol % cationic lipid/neutral lipid,e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA) or52/13/30/5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Non-limiting examples of lipid nanoparticle compositions and methods ofmaking them are described, for example, in Semple et al. (2010) Nat.Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed.,51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578(the contents of each of which are incorporated herein by reference intheir entirety).

In some embodiments, lipid nanoparticle formulations may comprise acationic lipid, a PEG lipid and a structural lipid and optionallycomprise a non-cationic lipid. As a non-limiting example, a lipidnanoparticle may comprise 40-60% of cationic lipid, 5-15% of anon-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structurallipid. As another non-limiting example, the lipid nanoparticle maycomprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and38.5% structural lipid. As yet another non-limiting example, a lipidnanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid,2.5% PEG lipid and 32.5% structural lipid. In some embodiments, thecationic lipid may be any cationic lipid described herein such as, butnot limited to, Dlin-KC2-DMA, Dlin-MC3-DMA and L319.

In some embodiments, the lipid nanoparticle formulations describedherein may be 4 component lipid nanoparticles. The lipid nanoparticlemay comprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticle maycomprise 40-60% of cationic lipid, 5-15% of a non-cationic lipid, 1-2%of a PEG lipid and 30-50% of a structural lipid. As another non-limitingexample, the lipid nanoparticle may comprise 50% cationic lipid, 10%non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid. As yetanother non-limiting example, the lipid nanoparticle may comprise 55%cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5%structural lipid. In some embodiments, the cationic lipid may be anycationic lipid described herein such as, but not limited to,Dlin-KC2-DMA, Dlin-MC3-DMA and L319.

In some embodiments, the lipid nanoparticle formulations describedherein may comprise a cationic lipid, a non-cationic lipid, a PEG lipidand a structural lipid. As a non-limiting example, the lipidnanoparticle comprise 50% of the cationic lipid Dlin-KC2-DMA, 10% of thenon-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of thestructural lipid cholesterol. As a non-limiting example, the lipidnanoparticle comprise 50% of the cationic lipid Dlin-MC3-DMA, 10% of thenon-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of thestructural lipid cholesterol. As a non-limiting example, the lipidnanoparticle comprise 50% of the cationic lipid Dlin-MC3-DMA, 10% of thenon-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DMG and 38.5% of thestructural lipid cholesterol. As yet another non-limiting example, thelipid nanoparticle comprise 55% of the cationic lipid L319, 10% of thenon-cationic lipid DSPC, 2.5% of the PEG lipid PEG-DMG and 32.5% of thestructural lipid cholesterol.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in a vaccinecomposition may vary, depending upon the identity, size, and/orcondition of the subject being treated and further depending upon theroute by which the composition is to be administered. For example, thecomposition may comprise between 0.1% and 99% (w/w) of the activeingredient. By way of example, the composition may comprise between 0.1%and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, atleast 80% (w/w) active ingredient.

In some embodiments, the RNA vaccine composition may comprise thepolynucleotide described herein, formulated in a lipid nanoparticlecomprising MC3, Cholesterol, DSPC and PEG2000-DMG, the buffer trisodiumcitrate, sucrose and water for injection. As a non-limiting example, thecomposition comprises: 2.0 mg/mL of drug substance (e.g.,polynucleotides encoding H10N8 influenza virus), 21.8 mg/mL of MC3, 10.1mg/mL of cholesterol, 5.4 mg/mL of DSPC, 2.7 mg/mL of PEG2000-DMG, 5.16mg/mL of trisodium citrate, 71 mg/mL of sucrose and 1.0 mL of water forinjection.

In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has amean diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm. In someembodiments, a nanoparticle (e.g., a lipid nanoparticle) has a meandiameter of 50-150 nm, 50-200 nm, 80-100 nm or 80-200 nm.

In one embodiment, the RNA vaccines of the invention may be formulatedin lipid nanoparticles having a diameter from about 10 to about 100 nmsuch as, but not limited to, about 10 to about 20 nm, about 10 to about30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 toabout 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm,about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 toabout 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm,about 50 to about 70 nm about 50 to about 80 nm, about 50 to about 90nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 toabout 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80to about 90 nm, about 80 to about 100 nm and/or about 90 to about 100nm.

In one embodiment, the lipid nanoparticles may have a diameter fromabout 10 to 500 nm.

In one embodiment, the lipid nanoparticle may have a diameter greaterthan 100 nm, greater than 150 nm, greater than 200 nm, greater than 250nm, greater than 300 nm, greater than 350 nm, greater than 400 nm,greater than 450 nm, greater than 500 nm, greater than 550 nm, greaterthan 600 nm, greater than 650 nm, greater than 700 nm, greater than 750nm, greater than 800 nm, greater than 850 nm, greater than 900 nm,greater than 950 nm or greater than 1000 nm.

Modes of Vaccine Administration

CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNAvaccines, may be administered by any route which results in atherapeutically effective outcome. These include, but are not limited,to intradermal, intramuscular, and/or subcutaneous administration. Thepresent disclosure provides methods comprising administering RNAvaccines to a subject in need thereof. The exact amount required willvary from subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the disease, the particularcomposition, its mode of administration, its mode of activity, and thelike. CHIKV, DENV and/or ZIKV RNA vaccines, including combination RNAvaccines, compositions are typically formulated in dosage unit form forease of administration and uniformity of dosage. It will be understood,however, that the total daily usage of CHIKV, DENV and/or ZIKV RNAvaccines, including combination RNA vaccines, compositions may bedecided by the attending physician within the scope of sound medicaljudgment. The specific therapeutically effective, prophylacticallyeffective, or appropriate imaging dose level for any particular patientwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

In certain embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, includingcombination RNA vaccines, may be administered at dosage levelssufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kgto 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kgto 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kgto 25 mg/kg, of subject body weight per day, one or more times a day, toobtain the desired therapeutic, diagnostic, prophylactic, or imagingeffect (see e.g., the range of unit doses described in InternationalPublication No WO2013078199, herein incorporated by reference in itsentirety). The desired dosage may be delivered three times a day, twotimes a day, once a day, every other day, every third day, every week,every two weeks, every three weeks, or every four weeks. In certainembodiments, the desired dosage may be delivered using multipleadministrations (e.g., two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, or more administrations). Whenmultiple administrations are employed, split dosing regimens such asthose described herein may be used.

In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, includingcombination RNA vaccines, may be administered at dosage levelssufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to 0.05mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05 mg/kgto 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5 mg/kgto 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1 mg/kgto 25 mg/kg, of subject body weight per day, one or more times a day,per week, per month, etc. to obtain the desired therapeutic, diagnostic,prophylactic, or imaging effect (see e.g., the range of unit dosesdescribed in International Publication No WO2013078199, hereinincorporated by reference in its entirety). The desired dosage may bedelivered three times a day, two times a day, once a day, every otherday, every third day, every week, every two weeks, every three weeks,every four weeks, every 2 months, every three months, every 6 months,etc. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used. In exemplaryembodiments, CHIKV, DENV and/or ZIKV RNA vaccines, including combinationRNA vaccines, may be administered at dosage levels sufficient to deliver0.0005 mg/kg to 0.01 mg/kg, e.g., about 0.0005 mg/kg to about 0.0075mg/kg, e.g., about 0.0005 mg/kg, about 0.001 mg/kg, about 0.002 mg/kg,about 0.003 mg/kg, about 0.004 mg/kg or about 0.005 mg/kg.

In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, includingcombination RNA vaccines, may be administered once or twice (or more) atdosage levels sufficient to deliver 0.025 mg/kg to 0.250 mg/kg, 0.025mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0mg/kg.

In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, includingcombination RNA vaccines, may be administered twice (e.g., Day 0 and Day7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 andDay 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9months later, Day 0 and 12 months later, Day 0 and 18 months later, Day0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 yearslater) at a total dose of or at dosage levels sufficient to deliver atotal dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0mg. Higher and lower dosages and frequency of administration areencompassed by the present disclosure. For example, a CHIKV, DENV and/orZIKV RNA vaccines, including combination RNA vaccines, may beadministered three or four times.

In some embodiments, CHIKV, DENV and/or ZIKV RNA vaccines, includingcombination RNA vaccines, may be administered twice (e.g., Day 0 and Day7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28, Day 0 and Day60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day 150, Day 0 andDay 180, Day 0 and 3 months later, Day 0 and 6 months later, Day 0 and 9months later, Day 0 and 12 months later, Day 0 and 18 months later, Day0 and 2 years later, Day 0 and 5 years later, or Day 0 and 10 yearslater) at a total dose of or at dosage levels sufficient to deliver atotal dose of 0.010 mg, 0.025 mg, 0.100 mg or 0.400 mg.

In some embodiments the RNA vaccine for use in a method of vaccinating asubject is administered the subject a single dosage of between 10 μg/kgand 400 μg/kg of the nucleic acid vaccine in an effective amount tovaccinate the subject. In some embodiments the RNA vaccine for use in amethod of vaccinating a subject is administered the subject a singledosage of between 10 μg and 400 μg of the nucleic acid vaccine in aneffective amount to vaccinate the subject.

A RNA vaccine pharmaceutical composition described herein can beformulated into a dosage form described herein, such as an intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal,and subcutaneous).

In some embodiments, a RNA (e.g., mRNA) vaccine for use in a method ofvaccinating a subject is administered the subject a single dosage of 10μg of the nucleic acid vaccine in an effective amount to vaccinate thesubject. In some embodiments, a RNA vaccine for use in a method ofvaccinating a subject is administered the subject a single dosage of 2μg of the nucleic acid vaccine in an effective amount to vaccinate thesubject. In some embodiments, a vaccine for use in a method ofvaccinating a subject is administered the subject two dosages of 10 μgof the nucleic acid vaccine in an effective amount to vaccinate thesubject. In some embodiments, a RNA vaccine for use in a method ofvaccinating a subject is administered the subject two dosages of 2 μg ofthe nucleic acid vaccine in an effective amount to vaccinate thesubject.

A RNA (e.g. mRNA) vaccine pharmaceutical composition described hereincan be formulated into a dosage form described herein, such as anintranasal, intratracheal, or injectable (e.g., intravenous,intraocular, intravitreal, intramuscular, intradermal, intracardiac,intraperitoneal, and subcutaneous).

RNA Vaccine Formulations and Methods of Use

Some aspects of the present disclosure provide formulations of a RNA(e.g., mRNA) vaccine, wherein the RNA vaccine is formulated in aneffective amount to produce an antigen specific immune response in asubject (e.g., production of antibodies specific to a CHIKV, DENV and/orZIKV antigenic polypeptide). “An effective amount” is a dose of an RNA(e.g., mRNA) vaccine effective to produce an antigen-specific immuneresponse. Also provided herein are methods of inducing anantigen-specific immune response in a subject.

In some embodiments, the antigen-specific immune response ischaracterized by measuring an anti-CHIKV, anti-DENV and/or anti-ZIKVantigenic polypeptide antibody titer produced in a subject administereda RNA (e.g., mRNA) vaccine as provided herein. An antibody titer is ameasurement of the amount of antibodies within a subject, for example,antibodies that are specific to a particular antigen or epitope of anantigen. Antibody titer is typically expressed as the inverse of thegreatest dilution that provides a positive result. Enzyme-linkedimmunosorbent assay (ELISA) is a common assay for determining antibodytiters, for example.

In some embodiments, an antibody titer is used to assess whether asubject has had an infection or to determine whether immunizations arerequired. In some embodiments, an antibody titer is used to determinethe strength of an autoimmune response, to determine whether a boosterimmunization is needed, to determine whether a previous vaccine waseffective, and to identify any recent or prior infections. In accordancewith the present disclosure, an antibody titer may be used to determinethe strength of an immune response induced in a subject by the RNAvaccine.

In some embodiments, an anti-ZIKV antigenic polypeptide antibody titerproduced in a subject is increased by at least 1 log relative to acontrol. For example, antibody titer produced in a subject may beincreased by at least 1.5, at least 2, at least 2.5, or at least 3 logrelative to a control. In some embodiments, the antibody titer producedin the subject is increased by 1, 1.5, 2, 2.5 or 3 log relative to acontrol. In some embodiments, the antibody titer produced in the subjectis increased by 1-3 log relative to a control. For example, the antibodytiter produced in a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3,1.5-2, 1.5-2.5, 1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control.

In some embodiments, the antibody titer produced in a subject isincreased at least 2 times relative to a control. For example, theantibody titer produced in a subject may be increased at least 3 times,at least 4 times, at least 5 times, at least 6 times, at least 7 times,at least 8 times, at least 9 times, or at least 10 times relative to acontrol. In some embodiments, the antibody titer produced in the subjectis increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 times relative to a control.In some embodiments, the anti antibody titer produced in a subject isincreased 2-10 times relative to a control. For example, the antibodytiter produced in a subject may be increased 2-10, 2-9, 2-8, 2-7, 2-6,2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7,4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8,8-10, 8-9, or 9-10 times relative to a control.

A control, in some embodiments, is an anti-CHIKV, anti-DENV and/oranti-ZIKV antigenic polypeptide antibody titer produced in a subject whohas not been administered a RNA (e.g., mRNA) vaccine. In someembodiments, a control is an anti-CHIKV, anti-DENV and/or anti-ZIKVantibody titer produced in a subject who has been administered a liveattenuated CHIKV, DENV and/or ZIKV vaccine. An attenuated vaccine is avaccine produced by reducing the virulence of a viable (live). Anattenuated virus is altered in a manner that renders it harmless or lessvirulent relative to live, unmodified virus. In some embodiments, acontrol is an anti-CHIKV, anti-DENV and/or anti-ZIKV antigenicpolypeptide antibody titer produced in a subject administeredinactivated CHIKV, DENV and/or ZIKV vaccine. In some embodiments, acontrol is an anti-CHIKV, anti-DENV and/or anti-ZIKV antigenicpolypeptide antibody titer produced in a subject administered arecombinant or purified CHIKV, DENV and/or ZIKV protein vaccine.Recombinant protein vaccines typically include protein antigens thateither have been produced in a heterologous expression system (e.g.,bacteria or yeast) or purified from large amounts of the pathogenicorganism.

In some embodiments, an effective amount of a RNA (e.g., mRNA) vaccineis a dose that is reduced compared to the standard of care dose of arecombinant CHIKV, DENV and/or ZIKV protein vaccine. A “standard ofcare,” as provided herein, refers to a medical or psychologicaltreatment guideline and can be general or specific. “Standard of care”specifies appropriate treatment based on scientific evidence andcollaboration between medical professionals involved in the treatment ofa given condition. It is the diagnostic and treatment process that aphysician/clinician should follow for a certain type of patient, illnessor clinical circumstance. A “standard of care dose,” as provided herein,refers to the dose of a recombinant or purified CHIKV, DENV and/or ZIKVprotein vaccine, or a live attenuated or inactivated CHIKV, DENV and/orZIKV vaccine, that a physician/clinician or other medical professionalwould administer to a subject to treat or prevent CHIKV, DENV and/orZIKV or a related condition, while following the standard of careguideline for treating or preventing CHIKV, DENV and/or ZIKV, or arelated condition.

In some embodiments, the anti-CHIKV, anti-DENV and/or anti-ZIKVantigenic polypeptide antibody titer produced in a subject administeredan effective amount of a ZIKV RNA vaccine is equivalent to ananti-CHIKV, anti-DENV and/or anti-ZIKV antigenic polypeptide antibodytiter produced in a control subject administered a standard of care doseof a recombinant or purified CHIKV, DENV and/or ZIKV protein vaccine ora live attenuated or inactivated CHIKV, DENV and/or ZIKV vaccine.

In some embodiments, an effective amount of a RNA (e.g., mRNA) vaccineis a dose equivalent to an at least 2-fold reduction in a standard ofcare dose of a recombinant or purified CHIKV, DENV and/or ZIKV proteinvaccine. For example, an effective amount of a CHIKV, DENV and/or ZIKVRNA vaccine may be a dose equivalent to an at least 3-fold, at least4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least8-fold, at least 9-fold, or at least 10-fold reduction in a standard ofcare dose of a recombinant or purified CHIKV, DENV and/or ZIKV proteinvaccine. In some embodiments, an effective amount of a CHIKV, DENVand/or ZIKV RNA vaccine is a dose equivalent to an at least at least100-fold, at least 500-fold, or at least 1000-fold reduction in astandard of care dose of a recombinant or purified CHIKV, DENV and/orZIKV protein vaccine. In some embodiments, an effective amount of aCHIKV, DENV and/or ZIKV RNA vaccine is a dose equivalent to a 2-, 3-,4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 50-, 100-, 250-, 500-, or 1000-foldreduction in a standard of care dose of a recombinant or purified CHIKV,DENV and/or ZIKV protein vaccine. In some embodiments, the anti-CHIKV,anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer producedin a subject administered an effective amount of a CHIKV, DENV and/orZIKV RNA vaccine is equivalent to an anti-CHIKV, anti-DENV and/oranti-ZIKV antigenic polypeptide antibody titer produced in a controlsubject administered the standard of care dose of a recombinant orprotein CHIKV, DENV and/or ZIKV protein vaccine or a live attenuated orinactivated CHIKV, DENV and/or ZIKV vaccine. In some embodiments, aneffective amount of a RNA (e.g., mRNA) vaccine is a dose equivalent to a2-fold to 1000-fold (e.g., 2-fold to 100-fold, 10-fold to 1000-fold)reduction in the standard of care dose of a recombinant or purifiedCHIKV, DENV and/or ZIKV protein vaccine, wherein the anti-CHIKV,anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer producedin the subject is equivalent to an anti-CHIKV, anti-DENV and/oranti-ZIKV antigenic polypeptide antibody titer produced in a controlsubject administered the standard of care dose of a recombinant orpurified CHIKV, DENV and/or ZIKV protein vaccine or a live attenuated orinactivated CHIKV, DENV and/or ZIKV vaccine.

In some embodiments, the effective amount of a RNA (e.g., mRNA) vaccineis a dose equivalent to a 2 to 1000-, 2 to 900-, 2 to 800-, 2 to 700-, 2to 600-, 2 to 500-, 2 to 400-, 2 to 300-, 2 to 200-, 2 to 100-, 2 to90-, 2 to 80-, 2 to 70-, 2 to 60-, 2 to 50-, 2 to 40-, 2 to 30-, 2 to20-, 2 to 10-, 2 to 9-, 2 to 8-, 2 to 7-, 2 to 6-, 2 to 5-, 2 to 4-, 2to 3-, 3 to 1000-, 3 to 900-, 3 to 800-, 3 to 700-, 3 to 600-, 3 to500-, 3 to 400-, 3 to 3 to 00-, 3 to 200-, 3 to 100-, 3 to 90-, 3 to80-, 3 to 70-, 3 to 60-, 3 to 50-, 3 to 40-, 3 to 30-, 3 to 20-, 3 to10-, 3 to 9-, 3 to 8-, 3 to 7-, 3 to 6-, 3 to 5-, 3 to 4-, 4 to 1000-, 4to 900-, 4 to 800-, 4 to 700-, 4 to 600-, 4 to 500-, 4 to 400-, 4 to 4to 00-, 4 to 200-, 4 to 100-, 4 to 90-, 4 to 80-, 4 to 70-, 4 to 60-, 4to 50-, 4 to 40-, 4 to 30-, 4 to 20-, 4 to 10-, 4 to 9-, 4 to 8-, 4 to7-, 4 to 6-, 4 to 5-, 4 to 4-, 5 to 1000-, 5 to 900-, 5 to 800-, 5 to700-, 5 to 600-, 5 to 500-, 5 to 400-, 5 to 300-, 5 to 200-, 5 to 100-,5 to 90-, 5 to 80-, 5 to 70-, 5 to 60-, 5 to 50-, 5 to 40-, 5 to 30-, 5to 20-, 5 to 10-, 5 to 9-, 5 to 8-, 5 to 7-, 5 to 6-, 6 to 1000-, 6 to900-, 6 to 800-, 6 to 700-, 6 to 600-, 6 to 500-, 6 to 400-, 6 to 300-,6 to 200-, 6 to 100-, 6 to 90-, 6 to 80-, 6 to 70-, 6 to 60-, 6 to 50-,6 to 40-, 6 to 30-, 6 to 20-, 6 to 10-, 6 to 9-, 6 to 8-, 6 to 7-, 7 to1000-, 7 to 900-, 7 to 800-, 7 to 700-, 7 to 600-, 7 to 500-, 7 to 400-,7 to 300-, 7 to 200-, 7 to 100-, 7 to 90-, 7 to 80-, 7 to 70-, 7 to 60-,7 to 50-, 7 to 40-, 7 to 30-, 7 to 20-, 7 to 10-, 7 to 9-, 7 to 8-, 8 to1000-, 8 to 900-, 8 to 800-, 8 to 700-, 8 to 600, 8 to 500-, 8 to 400-,8 to 300-, 8 to 200-, 8 to 100-, 8 to 90-, 8 to 80-, 8 to 70-, 8 to 60-,8 to 50-, 8 to 40-, 8 to 30-, 8 to 20-, 8 to 10-, 8 to 9-, 9 to 1000-, 9to 900-, 9 to 800-, 9 to 700-, 9 to 600-, 9 to 500-, 9 to 400-, 9 to300-, 9 to 200-, 9 to 100-, 9 to 90-, 9 to 80-, 9 to 70-, 9 to 60-, 9 to50-, 9 to 40-, 9 to 30-, 9 to 20-, 9 to 10-, 10 to 1000-, 10 to 900-, 10to 800-, 10 to 700-, 10 to 600-, 10 to 500-, 10 to 400-, 10 to 300-, 10to 200-, 10 to 100-, 10 to 90-, 10 to 80-, 10 to 70-, 10 to 60-, 10 to50-, 10 to 40-, 10 to 30-, 10 to 20-, 20 to 1000-, 20 to 900-, 20 to800-, 20 to 700-, 20 to 600-, 20 to 500-, 20 to 400-, 20 to 300-, 20 to200-, 20 to 100-, 20 to 90-, 20 to 80-, 20 to 70-, 20 to 60-, 20 to 50-,20 to 40-, 20 to 30-, 30 to 1000-, 30 to 900-, 30 to 800-, 30 to 700-,30 to 600-, 30 to 500-, 30 to 400-, 30 to 300-, 30 to 200-, 30 to 100-,30 to 90-, 30 to 80-, 30 to 70-, 30 to 60-, 30 to 50-, 30 to 40-, 40 to1000-, 40 to 900-, 40 to 800-, 40 to 700-, 40 to 600-, 40 to 500-, 40 to400-, 40 to 300-, 40 to 200-, 40 to 100-, 40 to 90-, 40 to 80-, 40 to70-, 40 to 60-, 40 to 50-, 50 to 1000-, 50 to 900-, 50 to 800-, 50 to700-, 50 to 600-, 50 to 500-, 50 to 400-, 50 to 300-, 50 to 200-, 50 to100-, 50 to 90-, 50 to 80-, 50 to 70-, 50 to 60-, 60 to 1000-, 60 to900-, 60 to 800-, 60 to 700-, 60 to 600-, 60 to 500-, 60 to 400-, 60 to300-, 60 to 200-, 60 to 100-, 60 to 90-, 60 to 80-, 60 to 70-, 70 to1000-, 70 to 900-, 70 to 800-, 70 to 700-, 70 to 600-, 70 to 500-, 70 to400-, 70 to 300-, 70 to 200-, 70 to 100-, 70 to 90-, 70 to 80-, 80 to1000-, 80 to 900-, 80 to 800-, 80 to 700-, 80 to 600-, 80 to 500-, 80 to400-, 80 to 300-, 80 to 200-, 80 to 100-, 80 to 90-, 90 to 1000-, 90 to900-, 90 to 800-, 90 to 700-, 90 to 600-, 90 to 500-, 90 to 400-, 90 to300-, 90 to 200-, 90 to 100-, 100 to 1000-, 100 to 900-, 100 to 800-,100 to 700-, 100 to 600-, 100 to 500-, 100 to 400-, 100 to 300-, 100 to200-, 200 to 1000-, 200 to 900-, 200 to 800-, 200 to 700-, 200 to 600-,200 to 500-, 200 to 400-, 200 to 300-, 300 to 1000-, 300 to 900-, 300 to800-, 300 to 700-, 300 to 600-, 300 to 500-, 300 to 400-, 400 to 1000-,400 to 900-, 400 to 800-, 400 to 700-, 400 to 600-, 400 to 500-, 500 to1000-, 500 to 900-, 500 to 800-, 500 to 700-, 500 to 600-, 600 to 1000-,600 to 900-, 600 to 800-, 600 to 700-, 700 to 1000-, 700 to 900-, 700 to800-, 800 to 1000-, 800 to 900-, or 900 to 1000-fold reduction in thestandard of care dose of a recombinant CHIKV, DENV and/or ZIKV proteinvaccine. In some embodiments, such as the foregoing, the anti-CHIKV,anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer producedin the subject is equivalent to an anti-CHIKV, anti-DENV and/oranti-ZIKV antigenic polypeptide antibody titer produced in a controlsubject administered the standard of care dose of a recombinant orpurified CHIKV, DENV and/or ZIKV protein vaccine or a live attenuated orinactivated CHIKV, DENV and/or ZIKV vaccine. In some embodiments, theeffective amount is a dose equivalent to (or equivalent to an at least)2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-,90-, 100-, 110-, 120-, 130-, 140-, 150-, 160-, 170-, 1280-, 190-, 200-,210-, 220-, 230-, 240-, 250-, 260-, 270-, 280-, 290-, 300-, 310-, 320-,330-, 340-, 350-, 360-, 370-, 380-, 390-, 400-, 410-, 420-, 430-, 440-,450-, 4360-, 470-, 480-, 490-, 500-, 510-, 520-, 530-, 540-, 550-, 560-,5760-, 580-, 590-, 600-, 610-, 620-, 630-, 640-, 650-, 660-, 670-, 680-,690-, 700-, 710-, 720-, 730-, 740-, 750-, 760-, 770-, 780-, 790-, 800-,810-, 820-, 830-, 840-, 850-, 860-, 870-, 880-, 890-, 900-, 910-, 920-,930-, 940-, 950-, 960-, 970-, 980-, 990-, or 1000-fold reduction in thestandard of care dose of a recombinant CHIKV, DENV and/or ZIKV proteinvaccine. In some embodiments, such as the foregoing, an anti-CHIKV,anti-DENV and/or anti-ZIKV antigenic polypeptide antibody titer producedin the subject is equivalent to an anti-CHIKV, anti-DENV and/oranti-ZIKV antigenic polypeptide antibody titer produced in a controlsubject administered the standard of care dose of a recombinant orpurified CHIKV, DENV and/or ZIKV protein vaccine or a live attenuated orinactivated CHIKV, DENV and/or ZIKV vaccine.

In some embodiments, the effective amount of a RNA (e.g., mRNA) vaccineis a total dose of 50-1000 μg. In some embodiments, the effective amountof a RNA (e.g., mRNA) vaccine is a total dose of 50-1000, 50-900,50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 50-90,50-80, 50-70, 50-60, 60-1000, 60-900, 60-800, 60-700, 60-600, 60-500,60-400, 60-300, 60-200, 60-100, 60-90, 60-80, 60-70, 70-1000, 70-900,70-800, 70-700, 70-600, 70-500, 70-400, 70-300, 70-200, 70-100, 70-90,70-80, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500, 80-400, 80-300,80-200, 80-100, 80-90, 90-1000, 90-900, 90-800, 90-700, 90-600, 90-500,90-400, 90-300, 90-200, 90-100, 100-1000, 100-900, 100-800, 100-700,100-600, 100-500, 100-400, 100-300, 100-200, 200-1000, 200-900, 200-800,200-700, 200-600, 200-500, 200-400, 200-300, 300-1000, 300-900, 300-800,300-700, 300-600, 300-500, 300-400, 400-1000, 400-900, 400-800, 400-700,400-600, 400-500, 500-1000, 500-900, 500-800, 500-700, 500-600,600-1000, 600-900, 600-900, 600-700, 700-1000, 700-900, 700-800,800-1000, 800-900, or 900-1000 μg. In some embodiments, the effectiveamount of a RNA (e.g., mRNA) vaccine is a total dose of 50, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950 or 1000 μg. In some embodiments, the effective amount is a doseof 25-500 μg administered to the subject a total of two times. In someembodiments, the effective amount of a RNA (e.g., mRNA) vaccine is adose of 25-500, 25-400, 25-300, 25-200, 25-100, 25-50, 50-500, 50-400,50-300, 50-200, 50-100, 100-500, 100-400, 100-300, 100-200, 150-500,150-400, 150-300, 150-200, 200-500, 200-400, 200-300, 250-500, 250-400,250-300, 300-500, 300-400, 350-500, 350-400, 400-500 or 450-500 μgadministered to the subject a total of two times. In some embodiments,the effective amount of a RNA (e.g., mRNA) vaccine is a total dose of25, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 μg administeredto the subject a total of two times.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

EXAMPLES Example 1 Manufacture of Polynucleotides

According to the present disclosure, the manufacture of polynucleotidesand or parts or regions thereof may be accomplished utilizing themethods taught in International Application WO2014/152027 entitled“Manufacturing Methods for Production of RNA Transcripts”, the contentsof which is incorporated herein by reference in its entirety.

Purification methods may include those taught in InternationalApplication WO2014/152030 and WO2014/152031, each of which isincorporated herein by reference in its entirety.

Detection and characterization methods of the polynucleotides may beperformed as taught in WO2014/144039, which is incorporated herein byreference in its entirety.

Characterization of the polynucleotides of the disclosure may beaccomplished using a procedure selected from the group consisting ofpolynucleotide mapping, reverse transcriptase sequencing, chargedistribution analysis, and detection of RNA impurities, whereincharacterizing comprises determining the RNA transcript sequence,determining the purity of the RNA transcript, or determining the chargeheterogeneity of the RNA transcript. Such methods are taught in, forexample, WO2014/144711 and WO2014/144767, the contents of each of whichis incorporated herein by reference in its entirety.

Example 2 Chimeric Polynucleotide Synthesis

Introduction

According to the present disclosure, two regions or parts of a chimericpolynucleotide may be joined or ligated using triphosphate chemistry.

According to this method, a first region or part of 100 nucleotides orless is chemically synthesized with a 5′ monophosphate and terminal3′desOH or blocked OH. If the region is longer than 80 nucleotides, itmay be synthesized as two strands for ligation.

If the first region or part is synthesized as a non-positionallymodified region or part using in vitro transcription (IVT), conversionthe 5′monophosphate with subsequent capping of the 3′ terminus mayfollow.

Monophosphate protecting groups may be selected from any of those knownin the art.

The second region or part of the chimeric polynucleotide may besynthesized using either chemical synthesis or IVT methods. IVT methodsmay include an RNA polymerase that can utilize a primer with a modifiedcap. Alternatively, a cap of up to 130 nucleotides may be chemicallysynthesized and coupled to the IVT region or part.

It is noted that for ligation methods, ligation with DNA T4 ligase,followed by treatment with DNAse should readily avoid concatenation.

The entire chimeric polynucleotide need not be manufactured with aphosphate-sugar backbone. If one of the regions or parts encodes apolypeptide, then it is preferable that such region or part comprise aphosphate-sugar backbone.

Ligation is then performed using any known click chemistry, orthoclickchemistry, solulink, or other bioconjugate chemistries known to those inthe art.

Synthetic Route

The chimeric polynucleotide is made using a series of starting segments.Such segments include:

(a) Capped and protected 5′ segment comprising a normal 3′OH (SEG. 1)

(b) 5′ triphosphate segment which may include the coding region of apolypeptide and comprising a normal 3′OH (SEG. 2)

(c) 5′ monophosphate segment for the 3′ end of the chimericpolynucleotide (e.g., the tail) comprising cordycepin or no 3′OH (SEG.3)

After synthesis (chemical or IVT), segment 3 (SEG. 3) is treated withcordycepin and then with pyrophosphatase to create the 5′monophosphate.

Segment 2 (SEG. 2) is then ligated to SEG. 3 using RNA ligase. Theligated polynucleotide is then purified and treated with pyrophosphataseto cleave the diphosphate. The treated SEG.2-SEG. 3 construct is thenpurified and SEG. 1 is ligated to the 5′ terminus. A furtherpurification step of the chimeric polynucleotide may be performed.

Where the chimeric polynucleotide encodes a polypeptide, the ligated orjoined segments may be represented as: 5′UTR (SEG. 1), open readingframe or ORF (SEG. 2) and 3′UTR+PolyA (SEG. 3).

The yields of each step may be as much as 90-95%.

Example 3 PCR for cDNA Production

PCR procedures for the preparation of cDNA are performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix12.5 μl; Forward Primer (10 μM) 0.75 μl; ReversePrimer (10 μM) 0.75 μl; Template cDNA −100 ng; and dH20 diluted to 25.0μl. The reaction conditions are at 95° C. for 5 min. and 25 cycles of98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45 sec, then72° C. for 5 min. then 4° C. to termination.

The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NANODROP™ andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 4 In Vitro Transcription (IVT)

The in vitro transcription reaction generates polynucleotides containinguniformly modified polynucleotides. Such uniformly modifiedpolynucleotides may comprise a region or part of the polynucleotides ofthe disclosure. The input nucleotide triphosphate (NTP) mix is madein-house using natural and un-natural NTPs.

A typical in vitro transcription reaction includes the following:

1 Template cDNA 1.0 μg 2 10x transcription buffer (400 mM 2.0 μlTris-HCl pH 8.0, 190 mM MgCl2, 50 mM DTT, 10 mM Spermidine) 3 CustomNTPs (25 mM each) 7.2 μl 4 RNase Inhibitor 20 U 5 T7 RNA polymerase 3000U 6 dH20 Up to 20.0 μl. and 7 Incubation at 37° C. for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase is then used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA is purifiedusing Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 5 Exemplary Nucleic Acids Encoding CHIKV E1 RNA Polynucleotidesfor Use in a RNA Vaccine

The following sequences are exemplary sequences that can be used toencode CHIKV E1 RNA polynucleotides for use in the CHIKV RNA vaccine:

TABLE 1 CHIKV E1 RNA polynucleotides SEQ ID Name Sequence NO ChiK.secE1TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA 1 HS3UPCRfreeATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGAC (CHIKVACCTGCACAGCTGTTGTTTCTGCTGCTGCTTTGGTTGCCCGATACCACCG secreted E1GTGACTACAAAGACGACGACGATAAATACGAGCACGTGACGGTAATACCA antigen)AACACTGTGGGGGTGCCATACAAGACCCTGGTAAATCGCCCAGGCTACTCTCCCATGGTGCTGGAGATGGAGCTCCAGTCTGTGACCTTAGAGCCAACCCTCTCACTCGACTATATCACCTGTGAATACAAAACAGTGATCCCATCCCCCTACGTGAAATGTTGCGGAACTGCAGAGTGTAAGGATAAGAGTCTGCCCGATTACAGCTGCAAGGTGTTTACAGGCGTGTATCCATTTATGTGGGGAGGAGCCTACTGTTTTTGCGATGCCGAAAATACTCAGCTGTCTGAAGCCCATGTGGAGAAGAGTGAAAGTTGCAAGACCGAATTTGCTAGTGCCTACAGGGCACACACCGCTTCTGCCTCCGCTAAACTCCGAGTCCTTTACCAGGGCAATAATATTACGGTCGCTGCCTACGCTAACGGGGACCACGCTGTGACAGTCAAGGACGCCAAATTCGTAGTGGGCCCAATGAGCTCCGCCTGGACTCCCTTCGACAACAAAATCGTCGTGTATAAAGGCGACGTGTACAATATGGACTACCCACCCTTCGGGGCTGGAAGACCGGGCCAGTTTGGAGATATCCAATCAAGGACACCCGAGTCAAAGGACGTGTACGCCAATACGCAGCTGGTGCTGCAGAGACCCGCCGCTGGTACCGTGCATGTGCCTTATTCCCAAGCTCCATCTGGCTTCAAGTACTGGTTGAAAGAGCGCGGTGCTTCGCTGCAGCATACAGCACCGTTCGGATGTCAGATAGCAACCAACCCTGTACGGGCTGTCAACTGTGCCGTGGGAAATATACCTATTTCCATCGACATTCCGGACGCAGCTTTCACACGTGTCGTTGATGCCCCCTCAGTGACTGACATGTCATGTGAGGTGCCTGCTTGCACCCACAGCAGCGATTTTGGCGGAGTGGCCATAATCAAGTACACCGCCTCCAAAAAAGGAAAGTGTGCCGTACACTCTATGACCAACGCCGTCACAATCAGAGAAGCCGACGTTGAAGTAGAGGGAAATTCACAGCTGCAAATCAGCTTCAGCACCGCTCTTGCCTCTGCTGAGTTTAGGGTTCAGGTTTGCAGTACTCAGGTGCACTGTGCAGCCGCTTGCCATCCCCCCAAGGATCATATCGTGAATTATCCTGCATCCCACACCACACTGGGAGTCCAGGATATCTCAACAACTGCAATGTCTTGGGTGCAGAAGATCACCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC Chik-TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA 2 Strain37997-ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGTACGA E1 (CHIKVACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAAGACTCTAG E1 antigen-TCAACAGACCGGGCTACAGCCCCATGGTATTGGAGATGGAGCTTCTGTCT StrainGTCACCTTGGAACCAACGCTATCGCTTGATTACATCACGTGCGAGTATAA 37997):AACCGTTATCCCGTCTCCGTACGTGAAATGCTGCGGTACAGCAGAGTGTAAGGACAAGAGCCTACCTGATTACAGCTGTAAGGTCTTCACCGGCGTCTACCCATTCATGTGGGGCGGCGCCTACTGCTTCTGCGACACCGAAAATACGCAATTGAGCGAAGCACATGTGGAGAAGTCCGAATCATGCAAAACAGAATTTGCATCAGCATACAGGGCTCATACCGCATCCGCATCAGCTAAGCTCCGCGTCCTTTACCAAGGAAATAATATCACTGTGGCTGCTTATGCAAACGGCGACCATGCCGTCACAGTTAAGGACGCTAAATTCATAGTGGGGCCAATGTCTTCAGCCTGGACACCTTTCGACAATAAAATCGTGGTGTACAAAGGCGACGTCTACAACATGGACTACCCGCCCTTCGGCGCAGGAAGACCAGGACAATTTGGCGACATCCAAAGTCGCACGCCTGAGAGCGAAGACGTCTATGCTAATACACAACTGGTACTGCAGAGACCGTCCGCGGGTACGGTGCACGTGCCGTACTCTCAGGCACCATCTGGCTTCAAGTATTGGCTAAAAGAACGAGGGGCGTCGCTGCAGCACACAGCACCATTTGGCTGTCAAATAGCAACAAACCCGGTAAGAGCGATGAACTGCGCCGTAGGGAACATGCCTATCTCCATCGACATACCGGACGCGGCCTTTACCAGGGTCGTCGACGCGCCATCTTTAACGGACATGTCGTGTGAGGTATCAGCCTGCACCCATTCCTCAGACTTTGGGGGCGTAGCCATCATTAAATATGCAGCCAGTAAGAAAGGCAAGTGTGCAGTGCACTCGATGACTAACGCCGTCACTATTCGGGAAGCTGAAATAGAAGTAGAAGGGAACTCTCAGTTGCAAATCTCTTTTTCGACGGCCCTAGCCAGCGCCGAATTTCGCGTACAAGTCTGTTCTACACAAGTACACTGTGCAGCCGAGTGCCATCCACCGAAAGACCATATAGTCAATTACCCGGCGTCACACACCACCCTCGGGGTCCAAGACATTTCCGCTACGGCGATGTCATGGGTGCAGAAGATCACGGGAGGTGTGGGACTGGTTGTCGCTGTTGCAGCACTGATCCTAATCGTGGTGCTATGCGTGTCGTTTAGCAGGCACTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC Chik-TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA 3 Strain37997-ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCTATG E1 (CHIKVGAACGAACAGCAGCCCCTGTTCTGGTTGCAGGCTCTTATCCCGCTGGCCG E1 antigen-CCTTGATCGTCCTGTGCAACTGTCTGAAACTCTTGCCATGCTGCTGTAAG StrainACCCTGGCTTTTTTAGCCGTAATGAGCATCGGTGCCCACACTGTGAGCGC 37997):GTACGAACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAAGACTCTTGTCAACAGACCGGGTTACAGCCCCATGGTGTTGGAGATGGAGCTACAATCAGTCACCTTGGAACCAACACTGTCACTTGACTACATCACGTGCGAGTACAAAACTGTCATCCCCTCCCCGTACGTGAAGTGCTGTGGTACAGCAGAGTGCAAGGACAAGAGCCTACCAGACTACAGCTGCAAGGTCTTTACTGGAGTCTACCCATTTATGTGGGGCGGCGCCTACTGCTTTTGCGACGCCGAAAATACGCAATTGAGCGAGGCACATGTAGAGAAATCTGAATCTTGCAAAACAGAGTTTGCATCGGCCTACAGAGCCCACACCGCATCGGCGTCGGCGAAGCTCCGCGTCCTTTACCAAGGAAACAACATTACCGTAGCTGCCTACGCTAACGGTGACCATGCCGTCACAGTAAAGGACGCCAAGTTTGTCGTGGGCCCAATGTCCTCCGCCTGGACACCTTTTGACAACAAAATCGTGGTGTACAAAGGCGACGTCTACAACATGGACTACCCACCTTTTGGCGCAGGAAGACCAGGACAATTTGGTGACATTCAAAGTCGTACACCGGAAAGTAAAGACGTTTATGCCAACACTCAGTTGGTACTACAGAGGCCAGCAGCAGGCACGGTACATGTACCATACTCTCAGGCACCATCTGGCTTCAAGTATTGGCTGAAGGAACGAGGAGCATCGCTACAGCACACGGCACCGTTCGGTTGCCAGATTGCGACAAACCCGGTAAGAGCTGTAAATTGCGCTGTGGGGAACATACCAATTTCCATCGACATACCGGATGCGGCCTTTACTAGGGTTGTCGATGCACCCTCTGTAACGGACATGTCATGCGAAGTACCAGCCTGCACTCACTCCTCCGACTTTGGGGGCGTCGCCATCATCAAATACACAGCTAGCAAGAAAGGTAAATGTGCAGTACATTCGATGACCAACGCCGTTACCATTCGAGAAGCCGACGTAGAAGTAGAGGGGAACTCCCAGCTGCAAATATCCTTCTCAACAGCCCTGGCAAGCGCCGAGTTTCGCGTGCAAGTGTGCTCCACACAAGTACACTGCGCAGCCGCATGCCACCCTCCAAAGGACCACATAGTCAATTACCCAGCATCACACACCACCCTTGGGGTCCAGGATATATCCACAACGGCAATGTCTTGGGTGCAGAAGATTACGGGAGGAGTAGGATTAATTGTTGCTGTTGCTGCCTTAATTTTAATTGTGGTGCTATGCGTGTCGTTTAGCAGGCACTAATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC chikv-TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA 4 Brazillian-ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGTACGA E1 (CHIKVACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAAGACTCTAG E1 antigen-TCAATAGACCGGGCTACAGTCCCATGGTATTGGAGATGGAACTACTGTCA BrazilianGTCACTTTGGAGCCAACGCTATCGCTTGATTACATCACGTGCGAGTACAA strain)AACCGTTATCCCGTCTCCGTACGTGAAATGCTGCGGTACAGCAGAGTGCAAGGACAAAAACCTACCTGACTACAGCTGTAAGGTCTTCACCGGCGTCTACCCATTTATGTGGGGCGGAGCCTACTGCTTCTGCGACGCTGAAAACACGCAATTGAGCGAAGCACACGTGGAGAAGTCCGAATCATGCAAAACAGAATTTGCATCAGCATACAGGGCTCATACCGCATCCGCATCAGCTAAGCTCCGCGTCCTTTACCAAGGAAATAACATCACTGTAACTGCCTATGCTAACGGCGACCATGCCGTCACAGTTAAGGACGCCAAATTCATTGTGGGGCCAATGTCTTCAGCCTGGACACCTTTCGACAACAAAATTGTGGTGTACAAAGGTGACGTCTATAACATGGACTACCCGCCCTTTGGCGCAGGAAGACCAGGACAATTTGGCGATATCCAAAGTCGCACACCTGAGAGTAAAGACGTCTATGCTAATACACAACTGGTACTGCAGAGACCGGCTGCGGGTACGGTACATGTGCCATACTCTCAGGCACCATCTGGCTTTAAGTATTGGCTAAAAGAACGAGGGGCGTCGCTGCAGCACACAGCACCATTTGGCTGCCAAATAGCAACAAACCCGGTAAGAGCGGTGAATTGCGCCGTAGGGAACATGCCCATCTCCATCGACATACCGGATGCGGCCTTCATTAGGGTCGTCGACGCGCCCTCTTTAACGGACATGTCGTGCGAGGTACCAGCCTGCACCCATTCCTCAGATTTCGGGGGCGTCGCCATTATTAAATATGCAGCCAGCAAGAAAGGCAAGTGTGCGGTGCATTCGATGACCAACGCCGTCACAATTCGGGAAGCTGAGATAGAAGTTGAAGGGAATTCTCAGCTGCAAATCTCTTTCTCGACGGCCTTGGCCAGCGCCGAATTCCGCGTACAAGTCTGTTCTACACAAGTACACTGTGTAGCCGAGTGCCACCCTCCGAAGGACCACATAGTCAATTACCCGGCGTCACATACCACCCTCGGGGTCCAGGACATTTCCGCTACGGCGCTGTCATGGGTGCAGAAGATCACGGGAGGCGTGGGACTGGTTGTCGCTGTTGCAGCACTGATTCTAATCGTGGTGCTATGCGTGTCGTTCAGCAGGCACTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC

Example 6 Exemplary Nucleic Acids Encoding CHIKV E2 RNA Polynucleotidesfor Use in a RNA Vaccine

The following sequences are exemplary sequences that can be used toencode CHIKV E2 RNA polynucleotides for use in a RNA vaccine:

TABLE 2 CHIKV E2 RNA polynucleotides SEQ ID Name Sequence NO ChiK.secE2ATGGAGACCCCAGCTCAGCTTCTGTTTCTTCTCCTTCTATGGCTGCCTGA 5 HS3UPCRfreeCACGACTGGACATCACCACCATCATCATAGTACAAAAGACAATTTCAATG (CHIKVTGTACAAGGCCACCCGCCCTTATTTAGCACACTGTCCAGATTGCGGTGAG secreted E2GGGCACTCCTGTCACTCTCCTATCGCCTTGGAGCGGATCCGGAATGAGGC antigen):GACCGATGGAACACTGAAAATCCAGGTAAGCTTGCAGATTGGCATCAAGACTGACGATAGCCATGATTGGACCAAACTACGGTATATGGATAGCCATACACCTGCCGATGCTGAACGGGCCGGTCTGCTTGTGAGAACTAGCGCTCCATGCACCATCACGGGGACAATGGGACATTTTATCCTGGCTAGATGCCCAAAGGGCGAAACCCTCACCGTCGGATTCACCGACTCAAGGAAAATTTCTCACACATGTACCCATCCCTTCCACCATGAGCCACCGGTGATCGGGCGCGAACGCTTCCACAGCAGGCCTCAGCATGGAAAAGAACTGCCATGCTCGACCTATGTACAGTCCACCGCCGCTACCGCCGAAGAGATCGAAGTGCATATGCCTCCCGACACACCCGACCGAACCCTAATGACACAACAATCTGGGAATGTGAAGATTACAGTCAATGGACAGACTGTGAGGTATAAGTGTAACTGCGGTGGCTCAAATGAGGGCCTCACCACAACGGATAAGGTGATCAATAACTGCAAAATTGACCAGTGTCACGCGGCCGTGACCAACCATAAGAACTGGCAGTACAACTCACCCTTAGTGCCTAGGAACGCTGAGCTGGGAGATCGCAAGGGGAAGATACACATTCCCTTCCCGTTGGCGAATGTGACCTGCCGTGTGCCAAAAGCGAGAAATCCTACCGTAACATATGGCAAAAATCAGGTGACCATGTTGCTCTACCCGGATCACCCAACTCTGCTGAGCTATCGGAATATGGGACAAGAACCCAATTACCACGAGGAATGGGTTACGCACAAGAAAGAGGTGACCCTTACAGTCCCTACTGAAGGTCTGGAAGTGACCTGGGGCAATAACGAGCCTTATAAGTACTGGCCCCAGATGAGTACAAACGGCACCGCCCATGGACATCCACACGAGATCATTCTGTATTACTACGAACTATATCCCACAATGACTGGCAAGCCTATACCAAACCCACTTCTCGGCCTTGATAGCACATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC chikv-TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA 6 Brazillian-ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGAGTAC E2 (CHIKVCAAGGACAACTTCAATGTCTATAAAGCCACAAGACCGTACTTAGCTCACT E2 antigen-GTCCCGACTGTGGAGAAGGGCACTCGTGCCATAGTCCCGTAGCATTAGAA BrazilianCGCATCAGAAATGAAGCGACAGACGGGACGCTGAAAATCCAGGTCTCCTT strain):GCAAATCGGAATAAAGACGGATGATAGCCACGATTGGACCAAGCTGCGTTACATGGACAACCACACGCCAGCGGACGCAGAGAGGGCGGGGCTATTTGTAAGAACATCAGCACCGTGCACGATTACTGGAACAATGGGACACTTCATCCTGACCCGATGTCCGAAAGGGGAAACTCTGACGGTGGGATTCACTGACAGTAGGAAGATCAGTCACTCATGTACGCACCCATTTCACCACGACCCTCCTGTGATAGGCCGGGAGAAATTCCATTCCCGACCGCAGCACGGTAAAGAGCTGCCTTGCAGCACGTACGTGCAGAGCACCGCCGCAACTACCGAGGAGATAGAGGTACACATGCCCCCAGACACCCCTGATCGCACATTGATGTCACAACAGTCCGGCAACGTAAAGATCACAGTTAATGGCCAGACGGTGCGGTACAAGTGTAATTGCGGTGGCTCAAATGAAGGACTAATAACTACAGACAAAGTGATTAATAACTGCAAAGTTGATCAATGTCATGCCGCGGTCACCAATCACAAAAAGTGGCAGTACAACTCCCCTCTGGTCCCGCGTAATGCTGAACTTGGGGACCGAAAAGGAAAAATCCACATCCCGTTTCCGCTGGCAAATGTAACATGCAGGGTGCCTAAAGCAAGGAACCCCACCGTGACGTACGGGAAAAACCAAGTCATCATGCTACTGTATCCCGACCACCCAACACTCCTGTCCTACCGGAACATGGGAGAAGAACCAAACTACCAAGAAGAGTGGGTGACGCATAAGAAGGAAGTCGTGCTAACCGTGCCGACTGAAGGGCTCGAGGTCACGTGGGGTAACAACGAGCCGTATAAGTATTGGCCGCAGTTATCTACAAACGGTACAGCCCATGGCCACCCGCATGAGATAATTCTGTATTATTATGAGCTGTACCCTACTATGACTGTAGTAGTTGTGTCAGTGGCCTCGTTCGTACTCCTGTCGATGGTGGGTGTGGCAGTGGGGATGTGCATGTGTGCACGACGCAGATGCATCACACCGTACGAACTGACACCAGGAGCTACCGTCCCTTTCCTGCTTAGCCTAATATGCTGCATCAGAACAGCTAAAGCGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC chikv-TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA 7 Brazillian-ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGAGTAT E2 (CHIKVTAAGGACCACTTCAATGTCTATAAAGCCACAAGACCGTACCTAGCTCACT E2 antigen-GTCCCGACTGTGGAGAAGGGCACTCGTGCCATAGTCCCGTAGCGCTAGAA BrazilianCGCATCAGAAACGAAGCGACAGACGGGACGTTGAAAATCCAGGTTTCCTT strain):GCAAATCGGAATAAAGACGGATGATAGCCATGATTGGACCAAGCTGCGTTATATGGACAATCACATGCCAGCAGACGCAGAGCGGGCCGGGCTATTTGTAAGAACGTCAGCACCGTGCACGATTACTGGAACAATGGGACACTTCATTCTGGCCCGATGTCCGAAAGGAGAAACTCTGACGGTGGGGTTCACTGACGGTAGGAAGATCAGTCACTCATGTACGCACCCATTTCACCATGACCCTCCTGTGATAGGCCGGGAAAAATTCCATTCCCGACCGCAGCACGGTAGGGAACTACCTTGCAGCACGTACGCGCAGAGCACCGCTGCAACTGCCGAGGAGATAGAGGTACACATGCCCCCAGACACCCCAGATCGCACATTAATGTCACAACAGTCCGGCAATGTAAAGATCACAGTCAATAGTCAGACGGTGCGGTACAAGTGCAATTGTGGTGACTCAAGTGAAGGATTAACCACTACAGATAAAGTGATTAATAACTGCAAGGTCGATCAATGCCATGCCGCGGTCACCAATCACAAAAAATGGCAGTATAACTCCCCTCTGGTCCCGCGTAATGCTGAATTCGGGGACCGGAAAGGAAAAGTTCACATTCCATTTCCTCTGGCAAATGTGACATGCAGGGTGCCTAAAGCAAGAAACCCCACCGTGACGTACGGAAAAAACCAAGTCATCATGTTGCTGTATCCTGACCACCCAACGCTCCTGTCCTACAGGAATATGGGAGAAGAACCAAACTATCAAGAAGAGTGGGTGACGCATAAGAAGGAGATCAGGTTAACCGTGCCGACTGAGGGGCTCGAGGTCACGTGGGGTAACAATGAGCCGTACAAGTATTGGCCGCAGTTATCCACAAACGGTACAGCCCACGGCCACCCGCATGAGATAATTCTGTATTATTATGAGCTGTACCCAACTATGACTGCGGTAGTTTTGTCAGTGGCCTCGTTCATACTCCTGTCGATGGTGGGTGTGGCAGTGGGGATGTGCATGTGTGCACGACGCAGATGCATTACACCGTACGAACTGACACCAGGAGCTACCGTCCCTTTCCTGCTTAGCCTAATATGCTGCATTAGAACAGCTAAAGCGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC Chik-StrainTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA 8 37997-E2ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCCATA (CHIKV E2TCTAGCTCATTGTCCTGACTGCGGAGAAGGGCATTCGTGCCACAGCCCTA Antigen-TCGCATTGGAGCGCATCAGAAATGAAGCAACGGACGGAACGCTGAAAATC StrainCAGGTCTCTTTGCAGATCGGGATAAAGACAGATGACAGCCACGATTGGAC 37997)CAAGCTGCGCTATATGGATAGCCATACGCCAGCGGACGCGGAGCGAGCCGGATTGCTTGTAAGGACTTCAGCACCGTGCACGATCACCGGGACCATGGGACACTTTATTCTCGCCCGATGCCCGAAAGGAGAGACGCTGACAGTGGGATTTACGGACAGCAGAAAGATCAGCCACACATGCACACACCCGTTCCATCATGAACCACCTGTGATAGGTAGGGAGAGGTTCCACTCTCGACCACAACATGGTAAAGAGTTACCTTGCAGCACGTACGTGCAGAGCACCGCTGCCACTGCTGAGGAGATAGAGGTGCATATGCCCCCAGATACTCCTGACCGCACGCTGATGACGCAGCAGTCTGGCAACGTGAAGATCACAGTTAATGGGCAGACGGTGCGGTACAAGTGCAACTGCGGTGGCTCAAACGAGGGACTGACAACCACAGACAAAGTGATCAATAACTGCAAAATTGATCAGTGCCATGCTGCAGTCACTAATCACAAGAATTGGCAATACAACTCCCCTTTAGTCCCGCGCAACGCTGAACTCGGGGACCGTAAAGGAAAGATCCACATCCCATTCCCATTGGCAAACGTGACTTGCAGAGTGCCAAAAGCAAGAAACCCTACAGTAACTTACGGAAAAAACCAAGTCACCATGCTGCTGTATCCTGACCATCCGACACTCTTGTCTTACCGTAACATGGGACAGGAACCAAATTACCACGAGGAGTGGGTGACACACAAGAAGGAGGTTACCTTGACCGTGCCTACTGAGGGTCTGGAGGTCACTTGGGGCAACAACGAACCATACAAGTACTGGCCGCAGATGTCTACGAACGGTACTGCTCATGGTCACCCACATGAGATAATCTTGTACTATTATGAGCTGTACCCCACTATGACTGTAGTCATTGTGTCGGTGGCCTCGTTCGTGCTTCTGTCGATGGTGGGCACAGCAGTGGGAATGTGTGTGTGCGCACGGCGCAGATGCATTACACCATATGAATTAACACCAGGAGCCACTGTTCCCTTCCTGCTCAGCCTGCTATGCTGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC

Example 7 Exemplary Nucleic Acids Encoding CHIKV E1-E2 RNAPolynucleotides for Use in a RNA Vaccine

The following sequences are exemplary sequences that can be used toencode CHIKV E1-E2 RNA polynucleotides for use in a RNA vaccine:

TABLE 3 CHIKV E1-E2 RNA polynucleotides SEQ ID Name Sequence NO chikv-TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA 9 Brazillian-ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGAGTAC E2-E1CAAGGACAACTTCAATGTCTATAAAGCCACAAGACCGTACTTAGCTCACT (CHIKV E1-GTCCCGACTGTGGAGAAGGGCACTCGTGCCATAGTCCCGTAGCATTAGAA E2 Antigen-CGCATCAGAAATGAAGCGACAGACGGGACGCTGAAAATCCAGGTCTCCTT BrazilianGCAAATCGGAATAAAGACGGATGATAGCCACGATTGGACCAAGCTGCGTT strain):ACATGGACAACCACACGCCAGCGGACGCAGAGAGGGCGGGGCTATTTGTAAGAACATCAGCACCGTGCACGATTACTGGAACAATGGGACACTTCATCCTGACCCGATGTCCGAAAGGGGAAACTCTGACGGTGGGATTCACTGACAGTAGGAAGATCAGTCACTCATGTACGCACCCATTTCACCACGACCCTCCTGTGATAGGCCGGGAGAAATTCCATTCCCGACCGCAGCACGGTAAAGAGCTGCCTTGCAGCACGTACGTGCAGAGCACCGCCGCAACTACCGAGGAGATAGAGGTACACATGCCCCCAGACACCCCTGATCGCACATTGATGTCACAACAGTCCGGCAACGTAAAGATCACAGTTAATGGCCAGACGGTGCGGTACAAGTGTAATTGCGGTGGCTCAAATGAAGGACTAATAACTACAGACAAAGTGATTAATAACTGCAAAGTTGATCAATGTCATGCCGCGGTCACCAATCACAAAAAGTGGCAGTACAACTCCCCTCTGGTCCCGCGTAATGCTGAACTTGGGGACCGAAAAGGAAAAATCCACATCCCGTTTCCGCTGGCAAATGTAACATGCAGGGTGCCTAAAGCAAGGAACCCCACCGTGACGTACGGGAAAAACCAAGTCATCATGCTACTGTATCCCGACCACCCAACACTCCTGTCCTACCGGAACATGGGAGAAGAACCAAACTACCAAGAAGAGTGGGTGACGCATAAGAAGGAAGTCGTGCTAACCGTGCCGACTGAAGGGCTCGAGGTCACGTGGGGTAACAACGAGCCGTATAAGTATTGGCCGCAGTTATCTACAAACGGTACAGCCCATGGCCACCCGCATGAGATAATTCTGTATTATTATGAGCTGTACCCTACTATGACTGTAGTAGTTGTGTCAGTGGCCTCGTTCGTACTCCTGTCGATGGTGGGTGTGGCAGTGGGGATGTGCATGTGTGCACGACGCAGATGCATCACACCGTACGAACTGACACCAGGAGCTACCGTCCCTTTCCTGCTTAGCCTAATATGCTGCATCAGAACAGCTAAAGCGTACGAACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAAGACTCTAGTCAATAGACCGGGCTACAGTCCCATGGTATTGGAGATGGAACTACTGTCAGTCACTTTGGAGCCAACGCTATCGCTTGATTACATCACGTGCGAGTACAAAACCGTTATCCCGTCTCCGTACGTGAAATGCTGCGGTACAGCAGAGTGCAAGGACAAAAACCTACCTGACTACAGCTGTAAGGTCTTCACCGGCGTCTACCCATTTATGTGGGGCGGAGCCTACTGCTTCTGCGACGCTGAAAACACGCAATTGAGCGAAGCACACGTGGAGAAGTCCGAATCATGCAAAACAGAATTTGCATCAGCATACAGGGCTCATACCGCATCCGCATCAGCTAAGCTCCGCGTCCTTTACCAAGGAAATAACATCACTGTAACTGCCTATGCTAACGGCGACCATGCCGTCACAGTTAAGGACGCCAAATTCATTGTGGGGCCAATGTCTTCAGCCTGGACACCTTTCGACAACAAAATTGTGGTGTACAAAGGTGACGTCTATAACATGGACTACCCGCCCTTTGGCGCAGGAAGACCAGGACAATTTGGCGATATCCAAAGTCGCACACCTGAGAGTAAAGACGTCTATGCTAATACACAACTGGTACTGCAGAGACCGGCTGCGGGTACGGTACATGTGCCATACTCTCAGGCACCATCTGGCTTTAAGTATTGGCTAAAAGAACGAGGGGCGTCGCTGCAGCACACAGCACCATTTGGCTGCCAAATAGCAACAAACCCGGTAAGAGCGGTGAATTGCGCCGTAGGGAACATGCCCATCTCCATCGACATACCGGATGCGGCCTTCATTAGGGTCGTCGACGCGCCCTCTTTAACGGACATGTCGTGCGAGGTACCAGCCTGCACCCATTCCTCAGATTTCGGGGGCGTCGCCATTATTAAATATGCAGCCAGCAAGAAAGGCAAGTGTGCGGTGCATTCGATGACCAACGCCGTCACAATTCGGGAAGCTGAGATAGAAGTTGAAGGGAATTCTCAGCTGCAAATCTCTTTCTCGACGGCCTTGGCCAGCGCCGAATTCCGCGTACAAGTCTGTTCTACACAAGTACACTGTGTAGCCGAGTGCCACCCTCCGAAGGACCACATAGTCAATTACCCGGCGTCACATACCACCCTCGGGGTCCAGGACATTTCCGCTACGGCGCTGTCATGGGTGCAGAAGATCACGGGAGGCGTGGGACTGGTTGTCGCTGTTGCAGCACTGATTCTAATCGTGGTGCTATGCGTGTCGTTCAGCAGGCACTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC chikv-TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA 10 Brazillian-ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGAGTAT E2-E1TAAGGACCACTTCAATGTCTATAAAGCCACAAGACCGTACCTAGCTCACT (CHIKV E1-GTCCCGACTGTGGAGAAGGGCACTCGTGCCATAGTCCCGTAGCGCTAGAA E2 Antigen-CGCATCAGAAACGAAGCGACAGACGGGACGTTGAAAATCCAGGTTTCCTT BrazilianGCAAATCGGAATAAAGACGGATGATAGCCATGATTGGACCAAGCTGCGTT strain):ATATGGACAATCACATGCCAGCAGACGCAGAGCGGGCCGGGCTATTTGTAAGAACGTCAGCACCGTGCACGATTACTGGAACAATGGGACACTTCATTCTGGCCCGATGTCCGAAAGGAGAAACTCTGACGGTGGGGTTCACTGACGGTAGGAAGATCAGTCACTCATGTACGCACCCATTTCACCATGACCCTCCTGTGATAGGCCGGGAAAAATTCCATTCCCGACCGCAGCACGGTAGGGAACTACCTTGCAGCACGTACGCGCAGAGCACCGCTGCAACTGCCGAGGAGATAGAGGTACACATGCCCCCAGACACCCCAGATCGCACATTAATGTCACAACAGTCCGGCAATGTAAAGATCACAGTCAATAGTCAGACGGTGCGGTACAAGTGCAATTGTGGTGACTCAAGTGAAGGATTAACCACTACAGATAAAGTGATTAATAACTGCAAGGTCGATCAATGCCATGCCGCGGTCACCAATCACAAAAAATGGCAGTATAACTCCCCTCTGGTCCCGCGTAATGCTGAATTCGGGGACCGGAAAGGAAAAGTTCACATTCCATTTCCTCTGGCAAATGTGACATGCAGGGTGCCTAAAGCAAGAAACCCCACCGTGACGTACGGAAAAAACCAAGTCATCATGTTGCTGTATCCTGACCACCCAACGCTCCTGTCCTACAGGAATATGGGAGAAGAACCAAACTATCAAGAAGAGTGGGTGACGCATAAGAAGGAGATCAGGTTAACCGTGCCGACTGAGGGGCTCGAGGTCACGTGGGGTAACAATGAGCCGTACAAGTATTGGCCGCAGTTATCCACAAACGGTACAGCCCACGGCCACCCGCATGAGATAATTCTGTATTATTATGAGCTGTACCCAACTATGACTGCGGTAGTTTTGTCAGTGGCCTCGTTCATACTCCTGTCGATGGTGGGTGTGGCAGTGGGGATGTGCATGTGTGCACGACGCAGATGCATTACACCGTACGAACTGACACCAGGAGCTACCGTCCCTTTCCTGCTTAGCCTAATATGCTGCATTAGAACAGCTAAAGCGTACGAACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAAGACTCTAGTCAACAGACCGGGCTACAGCCCCATGGTATTGGAGATGGAGCTTCTGTCTGTCACCTTGGAACCAACGCTATCGCTTGATTACATCACGTGCGAGTATAAAACCGTTATCCCGTCTCCGTACGTGAAATGCTGCGGTACAGCAGAGTGTAAGGACAAGAGCCTACCTGATTACAGCTGTAAGGTCTTCACCGGCGTCTACCCATTCATGTGGGGCGGCGCCTACTGCTTCTGCGACACCGAAAATACGCAATTGAGCGAAGCACATGTGGAGAAGTCCGAATCATGCAAAACAGAATTTGCATCAGCATACAGGGCTCATACCGCATCCGCATCAGCTAAGCTCCGCGTCCTTTACCAAGGAAATAATATCACTGTGGCTGCTTATGCAAACGGCGACCATGCCGTCACAGTTAAGGACGCTAAATTCATAGTGGGGCCAATGTCTTCAGCCTGGACACCTTTCGACAATAAAATCGTGGTGTACAAAGGCGACGTCTACAACATGGACTACCCGCCCTTCGGCGCAGGAAGACCAGGACAATTTGGCGACATCCAAAGTCGCACGCCTGAGAGCGAAGACGTCTATGCTAATACACAACTGGTACTGCAGAGACCGTCCGCGGGTACGGTGCACGTGCCGTACTCTCAGGCACCATCTGGCTTCAAGTATTGGCTAAAAGAACGAGGGGCGTCGCTGCAGCACACAGCACCATTTGGCTGTCAAATAGCAACAAACCCGGTAAGAGCGATGAACTGCGCCGTAGGGAACATGCCTATCTCCATCGACATACCGGACGCGGCCTTTACCAGGGTCGTCGACGCGCCATCTTTAACGGACATGTCGTGTGAGGTATCAGCCTGCACCCATTCCTCAGACTTTGGGGGCGTAGCCATCATTAAATATGCAGCCAGTAAGAAAGGCAAGTGTGCAGTGCACTCGATGACTAACGCCGTCACTATTCGGGAAGCTGAAATAGAAGTAGAAGGGAACTCTCAGTTGCAAATCTCTTTTTCGACGGCCCTAGCCAGCGCCGAATTTCGCGTACAAGTCTGTTCTACACAAGTACACTGTGCAGCCGAGTGCCATCCACCGAAAGACCATATAGTCAATTACCCGGCGTCACACACCACCCTCGGGGTCCAAGACATTTCCGCTACGGCGATGTCATGGGTGCAGAAGATCACGGGAGGTGTGGGACTGGTTGTCGCTGTTGCAGCACTGATCCTAATCGTGGTGCTATGCGTGTCGTTTAGCAGGCACATGAGTATTAAGGACCACTTCAATGTCTATAAAGCCACAAGACCGTACCTAGCTCACTGTCCCGACTGTGGAGAAGGGCACTCGTGCCATAGTCCCGTAGCGCTAGAACGCATCAGAAACGAAGCGACAGACGGGACGTTGAAAATCCAGGTTTCCTTGCAAATCGGAATAAAGACGGATGATAGCCATGATTGGACCAAGCTGCGTTATATGGACAATCACATGCCAGCAGACGCAGAGCGGGCCGGGCTATTTGTAAGAACGTCAGCACCGTGCACGATTACTGGAACAATGGGACACTTCATTCTGGCCCGATGTCCGAAAGGAGAAACTCTGACGGTGGGGTTCACTGACGGTAGGAAGATCAGTCACTCATGTACGCACCCATTTCACCATGACCCTCCTGTGATAGGCCGGGAAAAATTCCATTCCCGACCGCAGCACGGTAGGGAACTACCTTGCAGCACGTACGCGCAGAGCACCGCTGCAACTGCCGAGGAGATAGAGGTACACATGCCCCCAGACACCCCAGATCGCACATTAATGTCACAACAGTCCGGCAATGTAAAGATCACAGTCAATAGTCAGACGGTGCGGTACAAGTGCAATTGTGGTGACTCAAGTGAAGGATTAACCACTACAGATAAAGTGATTAATAACTGCAAGGTCGATCAATGCCATGCCGCGGTCACCAATCACAAAAAATGGCAGTATAACTCCCCTCTGGTCCCGCGTAATGCTGAATTCGGGGACCGGAAAGGAAAAGTTCACATTCCATTTCCTCTGGCAAATGTGACATGCAGGGTGCCTAAAGCAAGAAACCCCACCGTGACGTACGGAAAAAACCAAGTCATCATGTTGCTGTATCCTGACCACCCAACGCTCCTGTCCTACAGGAATATGGGAGAAGAACCAAACTATCAAGAAGAGTGGGTGACGCATAAGAAGGAGATCAGGTTAACCGTGCCGACTGAGGGGCTCGAGGTCACGTGGGGTAACAATGAGCCGTACAAGTATTGGCCGCAGTTATCCACAAACGGTACAGCCCACGGCCACCCGCATGAGATAATTCTGTATTATTATGAGCTGTACCCAACTATGACTGCGGTAGTTTTGTCAGTGGCCTCGTTCATACTCCTGTCGATGGTGGGTGTGGCAGTGGGGATGTGCATGTGTGCACGACGCAGATGCATTACACCGTACGAACTGACACCAGGAGCTACCGTCCCTTTCCTGCTTAGCCTAATATGCTGCATTAGAACAGCTAAAGCGTACGAACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAAGACTCTAGTCAACAGACCGGGCTACAGCCCCATGGTATTGGAGATGGAGCTTCTGTCTGTCACCTTGGAACCAACGCTATCGCTTGATTACATCACGTGCGAGTATAAAACCGTTATCCCGTCTCCGTACGTGAAATGCTGCGGTACAGCAGAGTGTAAGGACAAGAGCCTACCTGATTACAGCTGTAAGGTCTTCACCGGCGTCTACCCATTCATGTGGGGCGGCGCCTACTGCTTCTGCGACACCGAAAATACGCAATTGAGCGAAGCACATGTGGAGAAGTCCGAATCATGCAAAACAGAATTTGCATCAGCATACAGGGCTCATACCGCATCCGCATCAGCTAAGCTCCGCGTCCTTTACCAAGGAAATAATATCACTGTGGCTGCTTATGCAAACGGCGACCATGCCGTCACAGTTAAGGACGCTAAATTCATAGTGGGGCCAATGTCTTCAGCCTGGACACCTTTCGACAATAAAATCGTGGTGTACAAAGGCGACGTCTACAACATGGACTACCCGCCCTTCGGCGCAGGAAGACCAGGACAATTTGGCGACATCCAAAGTCGCACGCCTGAGAGCGAAGACGTCTATGCTAATACACAACTGGTACTGCAGAGACCGTCCGCGGGTACGGTGCACGTGCCGTACTCTCAGGCACCATCTGGCTTCAAGTATTGGCTAAAAGAACGAGGGGCGTCGCTGCAGCACACAGCACCATTTGGCTGTCAAATAGCAACAAACCCGGTAAGAGCGATGAACTGCGCCGTAGGGAACATGCCTATCTCCATCGACATACCGGACGCGGCCTTTACCAGGGTCGTCGACGCGCCATCTTTAACGGACATGTCGTGTGAGGTATCAGCCTGCACCCATTCCTCAGACTTTGGGGGCGTAGCCATCATTAAATATGCAGCCAGTAAGAAAGGCAAGTGTGCAGTGCACTCGATGACTAACGCCGTCACTATTCGGGAAGCTGAAATAGAAGTAGAAGGGAACTCTCAGTTGCAAATCTCTTTTTCGACGGCCCTAGCCAGCGCCGAATTTCGCGTACAAGTCTGTTCTACACAAGTACACTGTGCAGCCGAGTGCCATCCACCGAAAGACCATATAGTCAATTACCCGGCGTCACACACCACCCTCGGGGTCCAAGACATTTCCGCTACGGCGATGTCATGGGTGCAGAAGATCACGGGAGGTGTGGGACTGGTTGTCGCTGTTGCAGCACTGATCCTAATCGTGGTGCTATGCGTGTCGTTTAGCAGGCACTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC Chik-StrainTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA 11 37997-E2-E1ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGCCATA (CHIKV E1-TCTAGCTCATTGTCCTGACTGCGGAGAAGGGCATTCGTGCCACAGCCCTA E2 Antigen-TCGCATTGGAGCGCATCAGAAATGAAGCAACGGACGGAACGCTGAAAATC StrainCAGGTCTCTTTGCAGATCGGGATAAAGACAGATGACAGCCACGATTGGAC 37997):CAAGCTGCGCTATATGGATAGCCATACGCCAGCGGACGCGGAGCGAGCCGGATTGCTTGTAAGGACTTCAGCACCGTGCACGATCACCGGGACCATGGGACACTTTATTCTCGCCCGATGCCCGAAAGGAGAGACGCTGACAGTGGGATTTACGGACAGCAGAAAGATCAGCCACACATGCACACACCCGTTCCATCATGAACCACCTGTGATAGGTAGGGAGAGGTTCCACTCTCGACCACAACATGGTAAAGAGTTACCTTGCAGCACGTACGTGCAGAGCACCGCTGCCACTGCTGAGGAGATAGAGGTGCATATGCCCCCAGATACTCCTGACCGCACGCTGATGACGCAGCAGTCTGGCAACGTGAAGATCACAGTTAATGGGCAGACGGTGCGGTACAAGTGCAACTGCGGTGGCTCAAACGAGGGACTGACAACCACAGACAAAGTGATCAATAACTGCAAAATTGATCAGTGCCATGCTGCAGTCACTAATCACAAGAATTGGCAATACAACTCCCCTTTAGTCCCGCGCAACGCTGAACTCGGGGACCGTAAAGGAAAGATCCACATCCCATTCCCATTGGCAAACGTGACTTGCAGAGTGCCAAAAGCAAGAAACCCTACAGTAACTTACGGAAAAAACCAAGTCACCATGCTGCTGTATCCTGACCATCCGACACTCTTGTCTTACCGTAACATGGGACAGGAACCAAATTACCACGAGGAGTGGGTGACACACAAGAAGGAGGTTACCTTGACCGTGCCTACTGAGGGTCTGGAGGTCACTTGGGGCAACAACGAACCATACAAGTACTGGCCGCAGATGTCTACGAACGGTACTGCTCATGGTCACCCACATGAGATAATCTTGTACTATTATGAGCTGTACCCCACTATGACTGTAGTCATTGTGTCGGTGGCCTCGTTCGTGCTTCTGTCGATGGTGGGCACAGCAGTGGGAATGTGTGTGTGCGCACGGCGCAGATGCATTACACCATATGAATTAACACCAGGAGCCACTGTTCCCTTCCTGCTCAGCCTGCTATGCTGCCTATGGAACGAACAGCAGCCCCTGTTCTGGTTGCAGGCTCTTATCCCGCTGGCCGCCTTGATCGTCCTGTGCAACTGTCTGAAACTCTTGCCATGCTGCTGTAAGACCCTGGCTTTTTTAGCCGTAATGAGCATCGGTGCCCACACTGTGAGCGCGTACGAACACGTAACAGTGATCCCGAACACGGTGGGAGTACCGTATAAGACTCTTGTCAACAGACCGGGTTACAGCCCCATGGTGTTGGAGATGGAGCTACAATCAGTCACCTTGGAACCAACACTGTCACTTGACTACATCACGTGCGAGTACAAAACTGTCATCCCCTCCCCGTACGTGAAGTGCTGTGGTACAGCAGAGTGCAAGGACAAGAGCCTACCAGACTACAGCTGCAAGGTCTTTACTGGAGTCTACCCATTTATGTGGGGCGGCGCCTACTGCTTTTGCGACGCCGAAAATACGCAATTGAGCGAGGCACATGTAGAGAAATCTGAATCTTGCAAAACAGAGTTTGCATCGGCCTACAGAGCCCACACCGCATCGGCGTCGGCGAAGCTCCGCGTCCTTTACCAAGGAAACAACATTACCGTAGCTGCCTACGCTAACGGTGACCATGCCGTCACAGTAAAGGACGCCAAGTTTGTCGTGGGCCCAATGTCCTCCGCCTGGACACCTTTTGACAACAAAATCGTGGTGTACAAAGGCGACGTCTACAACATGGACTACCCACCTTTTGGCGCAGGAAGACCAGGACAATTTGGTGACATTCAAAGTCGTACACCGGAAAGTAAAGACGTTTATGCCAACACTCAGTTGGTACTACAGAGGCCAGCAGCAGGCACGGTACATGTACCATACTCTCAGGCACCATCTGGCTTCAAGTATTGGCTGAAGGAACGAGGAGCATCGCTACAGCACACGGCACCGTTCGGTTGCCAGATTGCGACAAACCCGGTAAGAGCTGTAAATTGCGCTGTGGGGAACATACCAATTTCCATCGACATACCGGATGCGGCCTTTACTAGGGTTGTCGATGCACCCTCTGTAACGGACATGTCATGCGAAGTACCAGCCTGCACTCACTCCTCCGACTTTGGGGGCGTCGCCATCATCAAATACACAGCTAGCAAGAAAGGTAAATGTGCAGTACATTCGATGACCAACGCCGTTACCATTCGAGAAGCCGACGTAGAAGTAGAGGGGAACTCCCAGCTGCAAATATCCTTCTCAACAGCCCTGGCAAGCGCCGAGTTTCGCGTGCAAGTGTGCTCCACACAAGTACACTGCGCAGCCGCATGCCACCCTCCAAAGGACCACATAGTCAATTACCCAGCATCACACACCACCCTTGGGGTCCAGGATATATCCACAACGGCAATGTCTTGGGTGCAGAAGATTACGGGAGGAGTAGGATTAATTGTTGCTGTTGCTGCCTTAATTTTAATTGTGGTGCTATGCGTGTCGTTTAGCAGGCACTAATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCG GC

Example 8 Exemplary Nucleic Acids Encoding CHIKV C-E3-E2-6K-E1 RNAPolynucleotides for Use in a RNA Vaccine

The following sequence is an exemplary sequence that can be used toencode an CHIKV, DENV and/or ZIKV RNA polynucleotide C-E3-E2-6K-E1 foruse in a RNA vaccine:

TABLE 4 CHIKV RNA polynucleotide C-E3-E2-6K-E1 SEQ ID Name Sequence NOChik.C-E3- TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA 12 E2-6K-ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGTT E1_HS3UPCRfreeTATCCCTACGCAGACGTTCTATAATCGGAGGTACCAGCCCAGGCCTTGGG (C-E3-CCCCCCGCCCTACAATCCAAGTGATAAGACCACGTCCCAGGCCGCAGAGA E2-6K-E1CAAGCCGGCCAATTGGCGCAACTCATCAGCGCAGTTAACAAGTTGACCAT Antigen)GCGAGCGGTTCCTCAGCAGAAGCCGAGGCGGAACCGGAAGAATAAGAAACAACGCCAAAAGAAACAGGCGCCGCAGAACGACCCTAAACAGAAGAAACAACCTCCCCAGAAAAAGCCAGCTCAGAAGAAGAAGAAGCCTGGACGCCGTGAAAGAATGTGCATGAAAATCGAAAATGATTGCATCTTTGAGGTGAAGCACGAGGGCAAAGTGATGGGGTACGCATGCCTGGTGGGCGATAAGGTCATGAAGCCAGCACATGTGAAGGGGACAATCGATAATGCTGATCTGGCCAAGCTAGCTTTTAAACGTAGCTCCAAATACGATCTTGAGTGTGCCCAGATACCTGTGCACATGAAATCTGATGCAAGCAAGTTCACACACGAGAAGCCTGAGGGCTATTATAACTGGCATCATGGTGCGGTTCAGTACTCCGGCGGCCGATTTACCATTCCTACAGGGGCAGGAAAGCCGGGCGATTCGGGGAGACCCATTTTCGACAACAAAGGCCGCGTGGTAGCTATCGTGCTCGGTGGGGCTAATGAGGGTGCACGTACTGCACTTAGCGTGGTTACCTGGAATAAGGACATTGTCACAAAGATTACACCGGAGGGAGCAGAGGAATGGAGCCTGGCACTGCCCGTTCTGTGCCTGCTGGCCAACACCACTTTCCCATGTAGTCAACCCCCTTGCACTCCCTGCTGCTATGAGAAAGAGCCTGAGAGCACGTTACGTATGCTGGAAGATAATGTCATGAGGCCCGGGTACTATCAACTGCTCAAGGCTAGTCTGACATGCTCGCCCCACAGGCAGCGCAGGTCCACGAAAGATAACTTCAACGTTTACAAGGCTACTAGGCCTTATTTGGCCCACTGTCCCGATTGCGGAGAGGGACATTCTTGTCATAGTCCTATTGCCTTGGAGCGAATCCGCAACGAGGCCACTGATGGAACCCTTAAGATTCAAGTATCTTTGCAGATTGGCATTAAGACAGATGATTCCCATGACTGGACAAAGCTTCGGTACATGGACTCACACACGCCTGCAGATGCTGAAAGGGCAGGGCTCTTGGTCAGGACCTCGGCCCCTTGTACAATTACCGGGACCATGGGCCACTTCATCCTTGCACGCTGCCCTAAGGGGGAGACGCTGACGGTGGGCTTTACTGACTCGCGTAAGATCTCACACACATGTACACACCCTTTCCACCACGAACCTCCAGTCATAGGGAGAGAGAGATTTCACTCTCGCCCACAGCATGGCAAAGAGCTGCCATGCTCCACATATGTCCAGAGCACTGCTGCTACCGCTGAAGAAATTGAGGTTCACATGCCACCCGATACACCAGACCGTACTCTGATGACCCAACAGAGCGGCAACGTGAAGATTACCGTAAATGGACAGACCGTGAGATATAAGTGCAACTGTGGTGGCTCCAATGAGGGCTTAACAACAACGGATAAGGTGATTAACAATTGCAAAATAGATCAGTGCCATGCCGCAGTGACCAATCACAAGAATTGGCAATACAACTCACCCCTAGTGCCGAGGAACGCAGAACTAGGCGACAGGAAAGGGAAAATCCATATACCCTTCCCCCTAGCAAATGTGACCTGCCGAGTGCCCAAGGCCAGAAACCCCACGGTTACTTACGGCAAGAACCAGGTGACGATGCTTTTGTACCCAGACCATCCCACCTTGCTCTCTTATAGAAACATGGGACAGGAGCCTAACTATCATGAGGAGTGGGTGACACACAAGAAAGAAGTCACCCTTACCGTGCCTACCGAAGGGCTTGAAGTCACCTGGGGCAACAACGAGCCTTACAAGTATTGGCCACAGATGTCCACAAACGGAACAGCCCACGGCCACCCGCACGAGATCATACTGTATTACTATGAGCTTTATCCCACAATGACTGTCGTAATTGTGAGCGTTGCCAGCTTCGTGTTGCTTTCAATGGTTGGCACTGCCGTGGGGATGTGCGTGTGTGCTAGGCGCCGCTGTATAACTCCTTATGAACTAACTCCAGGCGCCACCGTTCCTTTCCTGCTCTCACTACTGTGTTGTGTGCGCACAACAAAGGCTGCCACCTACTACGAAGCCGCCGCCTACTTATGGAATGAACAGCAGCCTCTCTTTTGGTTACAGGCGCTGATTCCTCTTGCTGCCCTGATCGTGCTATGCAACTGCCTCAAGCTGCTGCCCTGTTGTTGCAAGACCCTAGCTTTTCTCGCCGTGATGAGCATCGGGGCACATACAGTGTCCGCCTATGAGCACGTCACCGTTATCCCGAACACCGTCGGTGTGCCATATAAGACGTTAGTCAATCGACCCGGCTACTCTCCAATGGTGCTGGAAATGGAACTCCAGAGTGTGACACTGGAGCCAACCTTATCCCTCGATTATATTACCTGCGAATACAAGACCGTCATCCCTTCACCCTATGTCAAGTGCTGTGGGACCGCTGAATGCAAAGACAAGAGCTTGCCTGATTACAGTTGCAAGGTCTTCACAGGTGTGTACCCCTTCATGTGGGGGGGAGCTTATTGCTTTTGTGATGCTGAGAACACCCAACTGAGCGAGGCTCACGTCGAGAAATCTGAGTCTTGCAAGACCGAGTTTGCCTCAGCTTACAGGGCCCACACGGCCAGCGCATCCGCCAAATTGAGGGTACTCTACCAGGGTAATAATATCACCGTTGCCGCATATGCAAACGGCGATCACGCCGTGACTGTCAAGGATGCCAAGTTCGTTGTGGGCCCCATGTCTAGCGCTTGGACACCGTTCGATAATAAGATCGTCGTGTACAAAGGGGACGTGTATAATATGGACTACCCACCTTTCGGGGCCGGCCGACCGGGACAGTTCGGGGATATTCAGAGCCGCACACCCGAATCTAAAGATGTTTACGCCAATACTCAGCTCGTCCTGCAGAGGCCCGCCGCTGGTACAGTTCACGTTCCTTACTCACAGGCACCCTCTGGGTTTAAGTATTGGCTGAAAGAACGAGGTGCCAGCTTGCAGCATACAGCGCCTTTCGGATGCCAGATTGCCACTAACCCCGTACGGGCTGTCAACTGCGCGGTCGGCAATATTCCCATTAGCATTGATATCCCGGACGCAGCTTTCACCAGGGTTGTGGACGCCCCGAGCGTCACCGACATGAGTTGTGAGGTGCCAGCCTGCACGCATAGCAGTGATTTCGGCGGCGTCGCCATCATTAAATATACCGCAAGCAAGAAAGGCAAGTGCGCCGTCCACTCGATGACTAACGCCGTCACAATTCGGGAAGCCGATGTTGAGGTCGAAGGCAACTCCCAGCTGCAGATCAGCTTCTCTACTGCTCTTGCAAGCGCCGAGTTTCGAGTCCAGGTCTGCAGTACGCAGGTGCATTGTGCAGCTGCCTGCCATCCACCCAAAGATCATATTGTGAATTATCCGGCGTCACATACCACACTGGGGGTCCAGGATATTAGTACAACGGCGATGTCCTGGGTGCAGAAAATTACGGGAGGAGTGGGCTTAATTGTTGCCGTGGCGGCCCTGATCCTGATCGTTGTGCTGTGTGTTAGCTTCTCTAGGCATGACTATAAAGATGACGATGACAAATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC CHIKV C-E3-TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAA 13 E2-6K-E1ATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGTTTATCCCTACGCAGACGTTCTATAATCGGAGGTACCAGCCCAGGCCTTGGGCCCCCCGCCCTACAATCCAAGTGATAAGACCACGTCCCAGGCCGCAGAGACAAGCCGGCCAATTGGCGCAACTCATCAGCGCAGTTAACAAGTTGACCATGCGAGCGGTTCCTCAGCAGAAGCCGAGGCGGAACCGGAAGAATAAGAAACAACGCCAAAAGAAACAGGCGCCGCAGAACGACCCTAAACAGAAGAAACAACCTCCCCAGAAAAAGCCAGCTCAGAAGAAGAAGAAGCCTGGACGCCGTGAAAGAATGTGCATGAAAATCGAAAATGATTGCATCTTTGAGGTGAAGCACGAGGGCAAAGTGATGGGGTACGCATGCCTGGTGGGCGATAAGGTCATGAAGCCAGCACATGTGAAGGGGACAATCGATAATGCTGATCTGGCCAAGCTAGCTTTTAAACGTAGCTCCAAATACGATCTTGAGTGTGCCCAGATACCTGTGCACATGAAATCTGATGCAAGCAAGTTCACACACGAGAAGCCTGAGGGCTATTATAACTGGCATCATGGTGCGGTTCAGTACTCCGGCGGCCGATTTACCATTCCTACAGGGGCAGGAAAGCCGGGCGATTCGGGGAGACCCATTTTCGACAACAAAGGCCGCGTGGTAGCTATCGTGCTCGGTGGGGCTAATGAGGGTGCACGTACTGCACTTAGCGTGGTTACCTGGAATAAGGACATTGTCACAAAGATTACACCGGAGGGAGCAGAGGAATGGAGCCTGGCACTGCCCGTTCTGTGCCTGCTGGCCAACACCACTTTCCCATGTAGTCAACCCCCTTGCACTCCCTGCTGCTATGAGAAAGAGCCTGAGAGCACGTTACGTATGCTGGAAGATAATGTCATGAGGCCCGGGTACTATCAACTGCTCAAGGCTAGTCTGACATGCTCGCCCCACAGGCAGCGCAGGTCCACGAAAGATAACTTCAACGTTTACAAGGCTACTAGGCCTTATTTGGCCCACTGTCCCGATTGCGGAGAGGGACATTCTTGTCATAGTCCTATTGCCTTGGAGCGAATCCGCAACGAGGCCACTGATGGAACCCTTAAGATTCAAGTATCTTTGCAGATTGGCATTAAGACAGATGATTCCCATGACTGGACAAAGCTTCGGTACATGGACTCACACACGCCTGCAGATGCTGAAAGGGCAGGGCTCTTGGTCAGGACCTCGGCCCCTTGTACAATTACCGGGACCATGGGCCACTTCATCCTTGCACGCTGCCCTAAGGGGGAGACGCTGACGGTGGGCTTTACTGACTCGCGTAAGATCTCACACACATGTACACACCCTTTCCACCACGAACCTCCAGTCATAGGGAGAGAGAGATTTCACTCTCGCCCACAGCATGGCAAAGAGCTGCCATGCTCCACATATGTCCAGAGCACTGCTGCTACCGCTGAAGAAATTGAGGTTCACATGCCACCCGATACACCAGACCGTACTCTGATGACCCAACAGAGCGGCAACGTGAAGATTACCGTAAATGGACAGACCGTGAGATATAAGTGCAACTGTGGTGGCTCCAATGAGGGCTTAACAACAACGGATAAGGTGATTAACAATTGCAAAATAGATCAGTGCCATGCCGCAGTGACCAATCACAAGAATTGGCAATACAACTCACCCCTAGTGCCGAGGAACGCAGAACTAGGCGACAGGAAAGGGAAAATCCATATACCCTTCCCCCTAGCAAATGTGACCTGCCGAGTGCCCAAGGCCAGAAACCCCACGGTTACTTACGGCAAGAACCAGGTGACGATGCTTTTGTACCCAGACCATCCCACCTTGCTCTCTTATAGAAACATGGGACAGGAGCCTAACTATCATGAGGAGTGGGTGACACACAAGAAAGAAGTCACCCTTACCGTGCCTACCGAAGGGCTTGAAGTCACCTGGGGCAACAACGAGCCTTACAAGTATTGGCCACAGATGTCCACAAACGGAACAGCCCACGGCCACCCGCACGAGATCATACTGTATTACTATGAGCTTTATCCCACAATGACTGTCGTAATTGTGAGCGTTGCCAGCTTCGTGTTGCTTTCAATGGTTGGCACTGCCGTGGGGATGTGCGTGTGTGCTAGGCGCCGCTGTATAACTCCTTATGAACTAACTCCAGGCGCCACCGTTCCTTTCCTGCTCTCACTACTGTGTTGTGTGCGCACAACAAAGGCTGCCACCTACTACGAAGCCGCCGCCTACTTATGGAATGAACAGCAGCCTCTCTTTTGGTTACAGGCGCTGATTCCTCTTGCTGCCCTGATCGTGCTATGCAACTGCCTCAAGCTGCTGCCCTGTTGTTGCAAGACCCTAGCTTTTCTCGCCGTGATGAGCATCGGGGCACATACAGTGTCCGCCTATGAGCACGTCACCGTTATCCCGAACACCGTCGGTGTGCCATATAAGACGTTAGTCAATCGACCCGGCTACTCTCCAATGGTGCTGGAAATGGAACTCCAGAGTGTGACACTGGAGCCAACCTTATCCCTCGATTATATTACCTGCGAATACAAGACCGTCATCCCTTCACCCTATGTCAAGTGCTGTGGGACCGCTGAATGCAAAGACAAGAGCTTGCCTGATTACAGTTGCAAGGTCTTCACAGGTGTGTACCCCTTCATGTGGGGGGGAGCTTATTGCTTTTGTGATGCTGAGAACACCCAACTGAGCGAGGCTCACGTCGAGAAATCTGAGTCTTGCAAGACCGAGTTTGCCTCAGCTTACAGGGCCCACACGGCCAGCGCATCCGCCAAATTGAGGGTACTCTACCAGGGTAATAATATCACCGTTGCCGCATATGCAAACGGCGATCACGCCGTGACTGTCAAGGATGCCAAGTTCGTTGTGGGCCCCATGTCTAGCGCTTGGACACCGTTCGATAATAAGATCGTCGTGTACAAAGGGGACGTGTATAATATGGACTACCCACCTTTCGGGGCCGGCCGACCGGGACAGTTCGGGGATATTCAGAGCCGCACACCCGAATCTAAAGATGTTTACGCCAATACTCAGCTCGTCCTGCAGAGGCCCGCCGCTGGTACAGTTCACGTTCCTTACTCACAGGCACCCTCTGGGTTTAAGTATTGGCTGAAAGAACGAGGTGCCAGCTTGCAGCATACAGCGCCTTTCGGATGCCAGATTGCCACTAACCCCGTACGGGCTGTCAACTGCGCGGTCGGCAATATTCCCATTAGCATTGATATCCCGGACGCAGCTTTCACCAGGGTTGTGGACGCCCCGAGCGTCACCGACATGAGTTGTGAGGTGCCAGCCTGCACGCATAGCAGTGATTTCGGCGGCGTCGCCATCATTAAATATACCGCAAGCAAGAAAGGCAAGTGCGCCGTCCACTCGATGACTAACGCCGTCACAATTCGGGAAGCCGATGTTGAGGTCGAAGGCAACTCCCAGCTGCAGATCAGCTTCTCTACTGCTCTTGCAAGCGCCGAGTTTCGAGTCCAGGTCTGCAGTACGCAGGTGCATTGTGCAGCTGCCTGCCATCCACCCAAAGATCATATTGTGAATTATCCGGCGTCACATACCACACTGGGGGTCCAGGATATTAGTACAACGGCGATGTCCTGGGTGCAGAAAATTACGGGAGGAGTGGGCTTAATTGTTGCCGTGGCGGCCCTGATCCTGATCGTTGTGCTGTGTGTTAGCTTCTCTAGGCATTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGG GCGGC CHIKV C-E3-SSFWTLVQKLIRLTIGKERKEEEEIEPPWSLSLRRRSIIGGTSPGLGPPA 14 E2-6K-E1LQSKDHVPGRRDKPANWRNSSAQLTSPCERFLSRSRGGTGRIRNNAKRNRRRRTTLNRRNNLPRKSQLRRRRSLDAVKECAKSKMIASLRSTRAKWGTHAWWAIRSSQHMRGQSIMLIWPSLLNVAPNTILSVPRYLCTNLMQASSHTRSLRAIITGIMVRFSTPAADLPFLQGQESRAIRGDPFSTTKAAWLSCSVGLMRVHVLHLAWLPGIRTLSQRLHRREQRNGAWHCPFCACWPTPLSHVVNPLALPAAMRKSLRARYVCWKIMSGPGTINCSRLVHARPTGSAGPRKITSTFTRLLGLIWPTVPIAERDILVIVLLPWSESATRPLMEPLRFKYLCRLALRQMIPMTGQSFGTWTHTRLQMLKGQGSWSGPRPLVQLPGPWATSSLHAALRGRRRWALLTRVRSHTHVHTLSTTNLQSGERDFTLAHSMAKSCHAPHMSRALLLPLKKLRFTCHPIHQTVLPNRAATRLPMDRPDISATVVAPMRAQQRIRLTIAKISAMPQPITRIGNTTHPCRGTQNATGKGKSIYPSPQMPAECPRPETPRLLTARTRRCFCTQTIPPCSLIETWDRSLTIMRSGHTRKKSPLPCLPKGLKSPGATTSLTSIGHRCPQTEQPTATRTRSYCITMSFIPQLSLALPASCCFQWLALPWGCACVLGAAVLLMNLQAPPFLSCSHYCVVCAQQRLPPTTKPPPTYGMNSSLSFGYRRFLLLPSCYATASSCCPVVARPLFSPASGHIQCPPMSTSPLSRTPSVCHIRRSIDPATLQWCWKWNSRVHWSQPYPSIILPANTRPSSLHPMSSAVGPLNAKTRACLITVARSSQVCTPSCGGELIAFVMLRTPNARLTSRNLSLARPSLPQLTGPTRPAHPPNGYSTRVIISPLPHMQTAITPLSRMPSSLWAPCLALGHRSIIRSSCTKGTCIIWTTHLSGPADRDSSGIFRAAHPNLKMFTPILSSSCRGPPLVQFTFLTHRHPLGLSIGKNEVPACSIQRLSDARLPLTPYGLSTARSAIFPLALISRTQLSPGLWTPRASPTVVRCQPARIAVISAASPSLNIPQARKASAPSTRLTPSQFGKPMLRSKATPSCRSASLLLLQAPSFESRSAVRRCIVQLPAIHPKIILIIRRHIPHWGSRILVQRRCPGCRKLREEWALLPWRPSSLCCVLASLGIDNRLEPRWPCFLPLGPPPSPSSPSCT RTPVVFESLSGR

FIG. 2 shows a phylogenetic tree of chikungunya virus strains derivedfrom complete concatenated open reading frames for the nonstructural andstructural polyproteins. E1 amino acid substitutions that facilitated(Indian Ocean lineage) or prevented (Asian lineage) adaptation to Aedesalbopictus are shown on the right. CAR: Central African republic; ECSA:East/Central/South Africa

Example 9 Protocol to Determine Efficacy of mRNA-Encoded ChikungunyaAntigen Candidates Against CHIKV

Chikungunya has a polycistronic genome and different antigens, based onthe Chikungunya structural protein, are possible. There aremembrane-bound and secreted forms of E1 and E2, as well as the fulllength polyprotein antigen, which retains the protein's nativeconformation. Additionally, the different CHIKV genotypes can also yielddifferent antigens.

The efficacy of Chik candidate vaccines in AG129 mice against challengewith a lethal dose of CHIKV strain 181/25 was investigated. A129 mice,which lack IFN α/β receptor signaling, injected intradermally in thefootpad with 10⁴ PFU of CHIKV 181/25 virus have a 100% survival ratepost-injection. In contrast, AG129 mice, which lack IFN α/β and γreceptor signaling, injected intradermally in the footpad with 10⁴ PFUof CHIKV 181/25 virus do not survive past day 5 post-injection. Thetested vaccines included: MC3-LNP formulated mRNA encoded CHIKV-E1,MC3-LNP formulated mRNA encoded CHIKV-E2, and MC3-LNP formulated mRNAencoded CHIKV-E1/E2/E3/C. Fifteen groups of five AG129 mice werevaccinated via intradermal (ID) or intramuscular (IM) injection witheither 2 μg or 10 μg of the candidate vaccine. The vaccines were givento AG129 mice as single or two doses (second dose provided 28 days afterthe first dose). The positive control group was vaccinated viaintranasal instillation (20 μL volume) with heat-inactivated CHIKV.Phosphate-buffered saline (PBS) was used as a negative control.

On day 56, mice were challenged with 1×10⁴ PFU of CHIKV via ID injectionin 50μL volume and monitored for 10 days for weight loss, morbidity, andmortality. Mice that displayed severe illness, defined as >30% weightloss, a health score of 6 or above, extreme lethargy, and/or paralysiswere euthanized. Notably, mice “vaccinated” with heat-inactivated CHIKV(positive control group) became morbid and were euthanized following thesecond dose of HI-CHIKV (they were not included in the challenge portionof the study).

In addition, individual samples were tested for reactivity in asemi-quantitative ELISA for mouse IgG against eitherChikungunya-specific E1 (groups 1-4), Chikungunya-specific E2 (groups5-8), or Chikungunya-specific E1 and E2 proteins (groups 9-15).

The health status is scored as indicated in the following Table 5:

TABLE 5 Health Status SCORE INITIALS DESCRIPTION APPEARANCE MOBILITYATTITUDE 1 H Healthy Smooth Coat. Bright Eyes. Active, Scurrying,Burrowing Alert 2 SR Slightly Ruffled Slightly Ruffled coat (usuallyActive, Scurrying, Burrowing Alert only around head and neck) 3 RRuffled Ruffled Coat throughout Active, Scurrying, Burrowing Alert body.A “wet” appearance. 4 S Sick Very Ruffled coat. Slightly Walking, but noscurrying. Mildly closed, inset eyes. Lethargic 5 VS Very Sick VeryRuffled Coat. Closed, Slow to no movement. Will Extremely (Euthanize)inset eyes. return to upright position Lethargic if put on its side. 6 EEuthanize Very ruffled Coat. Closed, No movement or Completely inseteyes. Moribund Uncontrollable, spastic Unaware or in requiring humanemovements. Will NOT return to Noticeable euthanasia. upright position ifput on its Distress side. 7 D Deceased — — —

Example 10 Efficacy of Chikungunya E1 Antigen mRNA Vaccine Candidate

AG129 mice (n=5 per group) were vaccinated with 2 μg or 10 μg ofMC-3-LNP formulated mRNA encoding CHIKV E1. The AG129 mice werevaccinated on either Day 0 or Days 0 and 28 via IM or ID delivery. OnDay 56 following final vaccination all mice were challenged with alethal dose of CHIKV. The survival curve, percent weight loss, andhealth status of the mice vaccinated with 2 μg CHIKV E1 mRNA are shownin FIGS. 4A-C. The survival results are tabulated in Table 6 below. Thesurvival curve, percent weight loss, and health status of the micevaccinated with 10 μg CHIKV E1 mRNA are shown in FIGS. 8A-C. Thesurvival results are tabulated in Table 7 below.

TABLE 6 Survival of mice vaccinated with Chikungunya E1 antigen mRNA - 2μg dose E1 E1 E1 E1 days post IM LNP IM LNP ID LNP ID LNP infection Day0 Day 0, 28 Day 0 Day 0, 28 Vehicle 0.000 100.000 100.000 100.000100.000 100.000 4.000 80.000 40.000 40.000 60.000 5.000 0.000 0.0000.000 0.000 0.000

TABLE 7 Survival of mice vaccinated with Chikungunya E1 antigen mRNA -10 μg dose E1 E1 E1 E1 days post IM LNP IM LNP ID LNP ID LNP infectionDay 0 Day 0, 28 Day 0 Day 0, 28 Vehicle 0.000 100.000 100.000 100.000100.000 100.000 4.000 60.000 80.000 5.000 0.000 80.000 0.000 0.000 6.00060.000 80.000 10.000 60.000 80.000

As shown in Table 6, the 2 μg dose of CHIKV E1 mRNA vaccine gave noprotection post-CHIKV infection challenge when administered via IM or IDwith either a single dose or two doses. Likewise, the single dose of 10μg CHIKV E1 vaccine provided little to no protection when administeredvia IM or ID. However, as indicated in Table 7, the 10 μg dose of CHIKVE1 mRNA vaccine provided 60% protection post-CHIKV challenge whenadministered via IM using two doses and provided 80% protectionpost-CHIKV challenge when administered via ID using two doses.

In all experiments, the negative control mice had a ˜0% survival rate,as did the positive control mice (heat-inactivated CHIKV), which diedbefore CHIKV challenge. Some mice died during the vaccination period.

Example 11 Efficacy of Chikungunya E2 Antigen mRNA Vaccine Candidate

AG129 mice (n=5 per group) were vaccinated with 2 μg or 10 μg ofMC-3-LNP formulated mRNA encoding CHIKV E2. The mice were vaccinated oneither Day 0 or Days 0 and 28 via IM or ID delivery. On Day 56 followingfinal vaccination all mice were challenged with a lethal dose of CHIKV.The survival curve, percent weight loss, and health status of the micevaccinated with 2 μg CHIKV E2 mRNA are shown in FIGS. 5A-C. The survivalresults are tabulated in Table 8 below. The survival curve, percentweight loss, and health status of the mice vaccinated with 10 μg CHIKVE2 mRNA are shown in FIGS. 9A-C. The survival results are tabulated inTable 9 below.

TABLE 8 Survival of mice vaccinated with Chikungunya E2 antigen mRNA - 2μg dose E1 E1 E1 E1 days post IM LNP IM LNP ID LNP ID LNP infection Day0 Day 0, 28 Day 0 Day 0, 28 Vehicle 0.000 100.000 100.000 100.000100.000 100.000 3.000 60.000 4.000 20.000 80.000 0.000 5.000 0.000 0.0000.000 6.000 80.000 10.000 80.000 100.000

TABLE 9 Survival of mice vaccinated with Chikungunya E2 antigen mRNA -10 μg dose E2 E2 E2 E2 days post IM LNP IM LNP ID LNP ID LNP infectionDay 0 Day 0, 28 Day 0 Day 0, 28 Vehicle 0.000 100.000 100.000 100.000100.000 100.000 5.000 40.000 0.000 0.000 6.000 0.000 10.000 100.000100.000

As shown in Table 8, the 2 μg dose of CHIKV E2 mRNA vaccine gave noprotection post-CHIKV infection challenge when administered via IM or IDin a single dose. However, when provided in two doses, the 2 μg dose ofCHIKV E2 mRNA vaccine provided 80% protection when administered via IMand 100% protection when administered via ID post-CHIKV challenge. Asindicated in Table 9, the 10 μg dose of CHIKV E2 mRNA mouse provided noprotection post-CHIKV challenge when administered via IM or ID in asingle dose. However, administration of CHIKV E2 mRNA via IM or ID usingtwo doses provided 100% protection post-CHIKV challenge.

In all experiments, the negative control mice had a ˜0% survival rate,as did the positive control mice (heat-inactivated CHIKV) which diedprior to CHIKV challenge. Some mice died during the vaccination period.

Example 12 Efficacy of Chikungunya C-E3-E2-6K-E1 Antigen mRNA VaccineCandidate

AG129 mice (n=5 per group) were vaccinated with 2 μg or 10 μg ofMC-3-LNP formulated mRNA encoding CHIKV C-E3-E2-6K-E1 mRNA (SEQ IDNO:3). The AG129 mice were vaccinated on either Day 0 or Days 0 and 28via IM or ID delivery. On Day 56 following final vaccination all micewere challenged with a lethal dose of CHIKV. The survival curve, percentweight loss, and health status of the mice vaccinated with 2 μg CHIKVC-E3-E2-6K-E1 mRNA are shown in FIGS. 6A-C. The survival results aretabulated in Table 10 below. The survival curve, percent weight loss,and health status of the mice vaccinated with 10 μg CHIKVC-E3-E2-6K-E1/E2/E3/C mRNA are shown in FIGS. 10A-C. The survivalresults are tabulated in Table 11 below.

TABLE 10 Survival of mice vaccinated with Chikungunya C-E3-E2-6K-E1antigen mRNA - 2 μg E1/E2/ E1/E2/ E1/E2/ E1/E2/ E3C E3C E3C E3C dayspost IM LNP IM LNP ID LNP ID LNP infection Day 0 Day 0, 28 Day 0 Day 0,28 Vehicle 0.000 100.000 100.000 100.000 100.000 100.000 5.000 80.0000.000 10.000 100.000 100.000 80.000 100.000

TABLE 11 Survival of mice vaccinated with Chikungunya C-E3-E2-6K-E1antigen mRNA - 10 μg E1/E2/ E1/E2/ E1/E2/ E1/E2/ E3C E3C E3C E3C dayspost IM LNP IM LNP ID LNP ID LNP infection Day 0 Day 0, 28 Day 0 Day 0,28 Vehicle 0.000 100.000 100.000 100.000 100.000 100.000 5.000 0.00010.000 100.000 100.000 100.000 100.000

As shown in Table 10, the 2 μg dose of C-E3-E2-6K-E1 mRNA vaccineprovided 100% protection post-CHIKV challenge when administered via IMin a single dose and provided 80% protection post-CHIKV challenge whenadministered via ID in a single dose. The 2 μg dose of C-E3-E2-6K-E1mRNA vaccine provided 100% protection post-CHIKV challenge whenadministered via IM or ID in two doses. As shown in Table 11, the 10 μgdose of C-E3-E2-6K-E1 mRNA vaccine provided 100% protection post-CHIKVinfection challenge when administered via IM or ID in either a singledose or in two doses.

In all experiments, the negative control mice had a ˜0% survival rate,as did the positive control mice (heat-inactivated CHIKV) which diedprior to CHIKV challenge. Some mice died during the vaccination period.

Example 13 Summary of Survival Data Using Chikungunya Antigen mRNAVaccine Candidates CHIKV E1, CHIKV E2, and CHIKV C-E3-E2-6K-E1

Table 12 shows the survival data of the mice vaccinated with the CHIKVmRNA antigens used in the studies reported in Examples 10-12.

TABLE 12 Summary of Day 6 post-injection survival data Dose 10 Dose 2ug/mouse ug/mouse G# Antigen/route/regime (survival %) (survival %) 1Chik-E1-IM- single dose 0 0 2 Chik-E1-IM- two doses 60 0 3 Chik-E1-ID-single dose 0 0 4 Chik-E1-ID- two doses 80 0 5 Chik-E2-IM- single dose 00 6 Chik-E2-IM- two doses 100 80 7 Chik-E2-ID- single dose 0 0 8Chik-E2-ID- two doses 100 100 9 Chik-E1-E2-E3-C-6KIM- single dose 100100 10 Chik-E1-E2-E3-C-6KIM- two doses 100 100 11 Chik-E1-E2-E3-C-6KID-single dose 100 80 12 Chik-E1-E2-E3-C-6KID- two doses 100 100 13 HICHIKV (+) 0 0 14 HI CHIKV (+) 0 0 15 Control (−) 0 0

Example 14 In Vitro Transfection of mRNA-Encoded Chikungunya VirusEnvelope Protein

The in vitro transfection of mRNA encoding Notch and a PBS control wereperformed in 150 k HeLa cells/well transfected with 1 μg mRNA+2 μLLF2000/well in a 24 well plate. Lysate containing proteins expressedfrom the CHIKV envelope mRNAs transfected in HeLa cells were collected16 hours post-transfection and then detected by Western blotting with aV5 tag-HRP antibody. The successful detection of a CHIKV envelopeprotein is shown in FIG. 3.

Example 15 Detection of Immunity (Mouse IgG) Against EitherChikungunya-specific E1, Chikungunya-Specific E2, orChikungunya-Specific E1 and E2 Proteins

Serum samples from mice vaccinated with the CHIKV E1, E2, or E1-E2-E3-Cvaccine described in Examples 11-13 were tested using asemi-quantitative ELISA for the detection of mouse IgG against eitherChikungunya-specific E1, Chikungunya-specific E2, orChikungunya-specific E1 and E2 proteins.

Fifteen groups of five mice were vaccinated via intradermal (ID) orintramuscular (IM) injection with either 2 μg or 10 μg of the candidatevaccine. The vaccines were given to AG129 mice as single or two doses(second dose provided 28 days after the first dose). On day 56, micewere challenged with 1×104 PFU of CHIKV via ID injection in 50 μL volumeand monitored for 10 days for weight loss, morbidity, and mortality.Mice were bled on day 7 and day 28 post-vaccination via the peri-orbitalsinus (retro-orbital bleed). In addition, mice surviving the CHIKVchallenge were bled 10 days post-challenge.

The individual samples were tested for reactivity in a semi-quantitativeELISA for mouse IgG against either Chikungunya-specific E1,Chikungunya-specific E2, or Chikungunya-specific E1 and E2 proteins. Theresults are shown in FIGS. 50-52.

The data depicting the results of the ELISA assay to identify the amountof antibodies produced in AG129 mice in response to vaccination withmRNA encoding secreted CHIKV E1 structural protein, secreted CHIKV E2structural protein, or CHIKV full structural polyprotein C-E3-E2-6k-E1at a dose of 10 μg or 2 μg at 28 days post immunization is shown inFIGS. 50-51. The 10 μg of mRNA encoding CHIKV polyprotein producedsignificant levels of antibody in both studies. The data depicting acomparison of ELISA titers from the data of FIG. 50 to survival in thedata of FIG. 51 left panel is shown in FIG. 52. As shown in the survivalresults, the animals vaccinated with either dose (single or doubleadministration) of mRNA encoding CHIKV polyprotein had 100% survivalrates.

Example 16 Efficacy of Chikungunya Polyprotein (C-E3-E2-6K-E1) mRNAVaccine Candidate

AG129 mice (n=5 per group) were vaccinated with either 10 μg, 2 μg or0.4 μg of MC-3-LNP formulated mRNA encoded CHIKV polyprotein(C-E3-E2-6K-E1) (SEQ ID NO: 13). The mice were vaccinated on either Day0 or Days 0 and 28 via IM delivery. In one study, all mice werechallenged on day 56 with a lethal dose of CHIKV following finalvaccination. In another study, all mice were challenged on day 84 with alethal dose of CHIKV following final vaccination. The survival curve,percent weight loss, and health status of the mice vaccinated with 10μg, 2 μg or 0.4 μg mRNA were determined as described previously inExamples 10-12. The survival rates, neutralizing antibodies and bindingantibodies were assessed. Neutralizing antibodies were also identifiedagainst three different strains of CHIKV.

The survival rates of the mice vaccinated with mRNA encoding CHIKVC-E3-E2-6k-E1 is shown in FIG. 53. The data depicts vaccination at adose of 10 μg (left panels), 2 μg (middle panels) or 0.4 μg (rightpanels) at 56 days (top panels) or 112 days (bottom panels) postimmunization. These data demonstrate that a single 2 μg dose of the mRNAvaccine afforded 100% protection for at least 112 days (16 weeks.)Following the study out further, the data demonstrated that a single 2μg dose of the mRNA vaccine afforded 100% protection for at least 140days (20 weeks.)

The neutralizing antibody and binding antibody produced in treated miceis shown in FIGS. 54 and 55 respectively. As can be seen in FIGS. 54 and55, the levels of neutralizing Ab were dependent or dose and regimenwith the highest titers evident with 10 μg dosed twice (days 0 and 28).Plaque reduction neutralization tests (PRNT50 and PRNT80) were used toquantify the titer of neutralizing antibody for the virus. Antigenbinding Ab was determined by ELISA. The corresponding correlationbetween binding Ab and neutralizing antibodies is shown in the bottompanels of FIG. 55. Following the study out to 16 weeks showed that thehighest E1 titers were achieved when 10 μg mRNA vaccine was dosed twice.

The data depicting neutralizing antibodies against three differentstrains of CHIKV is shown in FIG. 56. The neutralizing antibodies weretested against three different strains of CHIKV, African-Senegal (leftpanel), La Reunion (middle panel) and CDC CAR (right panel). FIG. 56shows that the polyprotein-encoding mRNA vaccine elicited broadlyneutralizing antibodies against the three strains tested. Sera werefurther tested against Chik S27 strain (Chikungunya virus (strainS27-African prototype). The data depicting neutralizing antibodiesagainst CHIKV S27 strain is shown in FIG. 57. These data collectivelyshow that the polyprotein encoding mRNA vaccine elicited broadlyneutralizing antibodies against all four strains tested. The vaccineinduced neutralizing antibodies against multiple strains of Chikungunya.The prime and boost with the 10 μg dose produced the most robustneutralizing antibody response followed by the single dose with 10 μg.

Example 17 Transfection of mRNA Encoded CHIKV Structural Proteins

In vitro transfection of mRNA encoding CHIKV structural proteins and PBScontrol were performed in 400 k HeLa cells transfected with 1.25 ug mRNAlipoplexed with 5 ul LF2000/well in 6 well plate. Protein detection inHeLa cell lysate 16 h post transfection was measured. Lysates whichcontain proteins expressed from the CHIKV mRNAs transfected in HeLa werecollected 16 h post transfection. Proteins were detected by WB with antiFlag or and V5 antibody.

FIG. 12 show the results of the assay. mRNA encoded CHIKV structuralproteins. Protein production in the HeLa cell lysate 16 h posttransfection was detected.

Example 18 Exemplary Dengue Sequences

The following are nucleic acid (SEQ ID NO: 16, 18, 20, and 22) and aminoacid (SEQ ID NO: 15, 17, 19, and 21) sequences for each of DEN-1, DEN-2,DEN-3, and DEN-4.

TABLE 13 DENV polynucleotide sequences and amino acid sequences SEQ IDName Sequence NO DEN-1MNNQRKKTGRPSFNMLKRARNRVSTVSQLAKRFSKGLLSGQGPMKLVMAF 15 (NC_001477.1)IAFLRFLAIPPTAGILARWGSFKKNGAIKVLRGFKKEISNMLNIMNRRKRSVTMLLMLLPTALAFHLTTRGGEPHMIVSKQERGKSLLFKTSAGVNMCTLIAMDLGELCEDTMTYKCPRITETEPDDVDCWCNATETWVTYGTCSQTGEH RRDKRSVALAPHVGLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVIALFLAHAIGTSITQKGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCAKFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTTATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNEMVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLILKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNSRSTSLSMTCIAVGMVTLYLGVMVQADSGCVINWKGRELKCGSGIFVTNEVHTWTEQYKFQADSPKRLSAAIGKAWEEGVCGIRSATRLENIMWKQISNELNHILLENDMKFTVVVGDVSGILAQGKKMIRPQPMEHKYSWKSWGKAKIIGADVQNTTFIIDGPNTPECPDNQRAWNIWEVEDYGFGIFTTNIWLKLRDSYTQVCDHRLMSAAIKDSKAVHADMGYWIESEKNETWKLARASFIEVKTCIWPKSHTLWSNGVLESEMIIPKIYGGPISQHNYRPGYFTQTAGPWHLGKLELDFDLCEGTTVVVDEHCGNRGPSLRTTTVTGKTIHEWCCRSCTLPPLRFKGEDGCWYGMEIRPVKEKEENLVKSMVSAGSGEVDSFSLGLLCISIMIEEVMRSRWSRKMLMTGTLAVFLLLTMGQLTWNDLIRLCIMVGANASDKMGMGTTYLALMATFRMRPMFAVGLLFRRLTSREVLLLTVGLSLVASVELPNSLEELGDGLAMGIMMLKLLTDFQSHQLWATLLSLTFVKTTFSLHYAWKTMAMILSIVSLFPLCLSTTSQKTTWLPVLLGSLGCKPLTMFLITENKIWGRKSWPLNEGIMAVGIVSILLSSLLKNDVPLAGPLIAGGMLIACYVISGSSADLSLEKAAEVSWEEEAEHSGASHNILVEVQDDGTMKIKDEERDDTLTILLKATLLAISGVYPMSIPATLFVWYFWQKKKQRSGVLWDTPSPPEVERAVLDDGIYRILQRGLLGRSQVGVGVFQEGVFHTMWHVTRGAVLMYQGKRLEPSWASVKKDLISYGGGWRFQGSWNAGEEVQVIAVEPGKNPKNVQTAPGTFKTPEGEVGAIALDFKPGTSGSPIVNREGKIVGLYGNGVVTTSGTYVSAIAQAKASQEGPLPEIEDEVFRKRNLTIMDLHPGSGKTRRYLPAIVREAIKRKLRTLVLAPTRVVASEMAEALKGMPIRYQTTAVKSEHTGKEIVDLMCHATFTMRLLSPVRVPNYNMIIMDEAHFTDPASIAARGYISTRVGMGEAAAIFMTATPPGSVEAFPQSIQDEERDIPERSWNSGYDWITDFPGKTVWFVPSIKSGNDIANCLRKNGKRVVQLSRKTFDTEYQKTKNNDWDYVVTTDISEMGANFRADRVIDPRRCLKPVILKDGPERVILAGPMPVTVASAAQRRGRIGRNQNKEGDQYIYMGQPLNNDEDHAHWTEAKMLLDNINTPEGIIPALFEPEREKSAAIDGEYRLRGEARKTFVELMRRGDLPVWLSYKVASEGFQYSDRRWCFDGERNNQVLEENMDVEIWTKEGERKKLRPRWLDARTYSDPLALREFKEFAAGRRSVSGDLILEIGKLPQHLTQRAQNALDNLVMLHNSEQGGKAYRHAMEELPDTIETLMLLALIAVLTGGVTLFFLSGRGLGKTSIGLLCVIASSALLWMASVEPHWIAASIILEFFLMVLLIPEPDRQRTPQDNQLAYVVIGLLFMILTVAANEMGLLETTKKDLGIGHAAAENHHHAAMLDVDLHPASAWTLYAVATTIITPMMRHTIENTTANISLTAIANQAAILMGLDKGWPISKMDIGVPLLALGCYSQVNPLTLTAAVLMLVAHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGIVAIDLDPVVYDAKFEKQLGQIMLLILCTSQILLMRTTWALCESITLATGPLTTLWEGSPGKFWNTTIAVSMANIFRGSYLAGAGLAFSLMKSLGGGRRGTGAQGETLGEKWKRQLNQLSKSEFNTYKRSGIIEVDRSEAKEGLKRGETTKHAVSRGTAKLRWFVERNLVKPEGKVIDLGCGRGGWSYYCAGLKKVTEVKGYTKGGPGHEEPIPMATYGWNLVKLYSGKDVFFTPPEKCDTLLCDIGESSPNPTIEEGRTLRVLKMVEPWLRGNQFCIKILNPYMPSVVETLEQMQRKHGGMLVRNPLSRNSTHEMYWVSCGTGNIVSAVNMTSRMLLNRFTMAHRKPTYERDVDLGAGTRHVAVEPEVANLDIIGQRIENIKNEHKSTWHYDEDNPYKTWAYHGSYEVKPSGSASSMVNGVVRLLTKPWDVIPMVTQIAMTDTTPFGQQRVFKEKVDTRTPKAKRGTAQIMEVTARWLWGFLSRNKKPRICTREEFTRKVRSNAAIGAVFVDENQWNSAKEAVEDERFWDLVHRERELHKQGKCATCVYNMMGKREKKLGEFGKAKGSRAIWYMWLGARFLEFEALGFMNEDHWFSRENSLSGVEGEGLHKLGYILRDISKIPGGNMYADDTAGWDTRITEDDLQNEAKITDIMEPEHALLATSIFKLTYQNKVVRVQRPAKNGTVMDVISRRDQRGSGQVGTYGLNTFTNMEAQLIRQMESEGIFSPSELETPNLAERVLDWLKKHGTERLKRMAISGDDCVVKPIDDRFATALTALNDMGKVRKDIPQWEPSKGWNDWQQVPFCSHHFHQLIMKDGREIVVPCRNQDELVGRARVSQGAGWSLRETACLGKSYAQMWQLMYFHRRDLRLAANAI CSAVPVDWVPTSRTTWSIHAHHQWMTTEDMLSVWNRVWIEENPWMEDKTHVSSWEDVPYLGKREDQWCGSLIGLTARATWATNIQVAINQVRRLIGNENYLDFMTSMKRFKNESDPEGALW DEN-1agttgttagtctacgtggaccgacaagaacagtttcgaatcggaagcttg 16 (NC_001477.1)cttaacgtagttctaacagttttttattagagagcagatctctgatgaacaaccaacggaaaaagacgggtcgaccgtctttcaatatgctgaaacgcgcgagaaaccgcgtgtcaactgtttcacagttggcgaagagattctcaaaaggattgctttcaggccaaggacccatgaaattggtgatggcttttatagcattcctaagatttctagccatacctccaacagcaggaattttggctagatggggctcattcaagaagaatggagcgatcaaagtgttacggggtttcaagaaagaaatctcaaacatgttgaacataatgaacaggaggaaaagatctgtgaccatgctcctcatgctgctgcccacagccctggcgttccatctgaccacccgagggggagagccgcacatgatagttagcaagcaggaaagaggaaaatcacttttgtttaagacctctgcaggtgtcaacatgtgcacccttattgcaatggatttgggagagttatgtgaggacacaatgacctacaaatgcccccggatcactgagacggaaccagatgacgttgactgttggtgcaatgccacggagacatgggtgacctatggaacatgttctcaaactggtgaacaccgacgagacaaacgttccgtcgcactggcaccacacgtagggcttggtctagaaacaagaaccgaaacgtggatgtcctctgaaggcgcttggaaacaaatacaaaaagtggagacctgggctctgagacacccaggattcacggtgatagccctttttctagcacatgccataggaacatccatcacccagaaagggatcatttttattttgctgatgctggtaactccatccatggccatgcggtgcgtgggaataggcaacagagacttcgtggaaggactgtcaggagctacgtgggtggatgtggtactggagcatggaagttgcgtcactaccatggcaaaagacaaaccaacactggacattgaactcttgaagacggaggtcacaaaccctgccgtcctgcgcaaactgtgcattgaagctaaaatatcaaacaccaccaccgattcgagatgtccaacacaaggagaagccacgctggtggaagaacaggacacgaactttgtgtgtcgacgaacgttcgtggacagaggctggggcaatggttgtgggctattcggaaaaggtagcttaataacgtgtgctaagtttaagtgtgtgacaaaactggaaggaaagatagtccaatatgaaaacttaaaatattcagtgatagtcaccgtacacactggagaccagcaccaagttggaaatgagaccacagaacatggaacaactgcaaccataacacctcaagctcccacgtcggaaatacagctgacagactacggagctctaacattggattgttcacctagaacagggctagactttaatgagatggtgttgttgacaatgaaaaaaaaatcatggctcgtccacaaacaatggtttctagacttaccactgccttggacctcgggggcttcaacatcccaagagacttggaatagacaagacttgctggtcacatttaagacagctcatgcaaaaaagcaggaagtagtcgtactaggatcacaagaaggagcaatgcacactgcgttgactggagcgacagaaatccaaacgtctggaacgacaacaatttttgcaggacacctgaaatgcagattaaaaatggataaactgattttaaaagggatgtcatatgtaatgtgcacagggtcattcaagttagagaaggaagtggctgagacccagcatggaactgttctagtgcaggttaaatacgaaggaacagatgcaccatgcaagatccccttctcgtcccaagatgagaagggagtaacccagaatgggagattgataacagccaaccccatagtcactgacaaagaaaaaccagtcaacattgaagcggagccaccttttggtgagagctacattgtggtaggagcaggtgaaaaagctttgaaactaagctggttcaagaagggaagcagtatagggaaaatgtttgaagcaactgcccgtggagcacgaaggatggccatcctgggagacactgcatgggacttcggttctataggaggggtgttcacgtctgtgggaaaactgatacaccagatttttgggactgcgtatggagttttgttcagcggtgtttcttggaccatgaagataggaatagggattctgctgacatggctaggattaaactcaaggagcacgtccctttcaatgacgtgtatcgcagttggcatggtcacactgtacctaggagtcatggttcaggcggactcgggatgtgtaatcaactggaaaggcagagaactcaaatgtggaagcggcatttttgtcaccaatgaagtccacacctggacagagcaatataaattccaggccgactcccctaagagactatcagcggccattgggaaggcatgggaggagggtgtgtgtggaattcgatcagccactcgtctcgagaacatcatgtggaagcaaatatcaaatgaattaaaccacatcttacttgaaaatgacatgaaatttacagtggtcgtaggagacgttagtggaatcttggcccaaggaaagaaaatgattaggccacaacccatggaacacaaatactcgtggaaaagctggggaaaagccaaaatcataggagcagatgtacagaataccaccttcatcatcgacggcccaaacaccccagaatgccctgataaccaaagagcatggaacatttgggaagttgaagactatggatttggaattttcacgacaaacatatggttgaaattgcgtgactcctacactcaagtgtgtgaccaccggctaatgtcagctgccatcaaggatagcaaagcagtccatgctgacatggggtactggatagaaagtgaaaagaacgagacttggaagttggcaagagcctccttcatagaagttaagacatgcatctggccaaaatcccacactctatggagcaatggagtcctggaaagtgagatgataatcccaaagatatatggaggaccaatatctcagcacaactacagaccaggatatttcacacaaacagcagggccgtggcacttgggcaagttagaactagattttgatttatgtgaaggtaccactgttgttgtggatgaacattgtggaaatcgaggaccatctcttagaaccacaacagtcacaggaaagacaatccatgaatggtgctgtagatcttgcacgttaccccccctacgtttcaaaggagaagacgggtgctggtacggcatggaaatcagaccagtcaaggagaaggaagagaacctagttaagtcaatggtctctgcagggtcaggagaagtggacagtttttcactaggactgctatgcatatcaataatgatcgaagaggtaatgagatccagatggagcagaaaaatgctgatgactggaacattggctgtgttcctccttctcacaatgggacaattgacatggaatgatctgatcaggctatgtatcatggttggagccaacgcttcagacaagatggggatgggaacaacgtacctagctttgatggccactttcagaatgagaccaatgttcgcagtcgggctactgtttcgcagattaacatctagagaagttcttcttcttacagttggattgagtctggtggcatctgtagaactaccaaattccttagaggagctaggggatggacttgcaatgggcatcatgatgttgaaattactgactgattttcagtcacatcagctatgggctaccttgctgtctttaacatttgtcaaaacaactttttcattgcactatgcatggaagacaatggctatgatactgtcaattgtatctctcttccctttatgcctgtccacgacttctcaaaaaacaacatggcttccggtgttgctgggatctcttggatgcaaaccactaaccatgtttcttataacagaaaacaaaatctggggaaggaaaagctggcctctcaatgaaggaattatggctgttggaatagttagcattcttctaagttcacttctcaagaatgatgtgccactagctggcccactaatagctggaggcatgctaatagcatgttatgtcatatctggaagctcggccgatttatcactggagaaagcggctgaggtctcctgggaagaagaagcagaacactctggtgcctcacacaacatactagtggaggtccaagatgatggaaccatgaagataaaggatgaagagagagatgacacactcaccattctcctcaaagcaactctgctagcaatctcaggggtatacccaatgtcaataccggcgaccctctttgtgtggtatttttggcagaaaaagaaacagagatcaggagtgctatgggacacacccagccctccagaagtggaaagagcagtccttgatgatggcatttatagaattctccaaagaggattgttgggcaggtctcaagtaggagtaggagtttttcaagaaggcgtgttccacacaatgtggcacgtcaccaggggagctgtcctcatgtaccaagggaagagactggaaccaagttgggccagtgtcaaaaaagacttgatctcatatggaggaggttggaggtttcaaggatcctggaacgcgggagaagaagtgcaggtgattgctgttgaaccggggaagaaccccaaaaatgtacagacagcgccgggtaccttcaagacccctgaaggcgaagttggagccatagctctagactttaaacccggcacatctggatctcctatcgtgaacagagagggaaaaatagtaggtctttatggaaatggagtggtgacaacaagtggtacctacgtcagtgccatagctcaagctaaagcatcacaagaagggcctctaccagagattgaggacgaggtgtttaggaaaagaaacttaacaataatggacctacatccaggatcgggaaaaacaagaagataccttccagccatagtccgtgaggccataaaaagaaagctgcgcacgctagtcttagctcccacaagagttgtcgcttctgaaatggcagaggcgctcaagggaatgccaataaggtatcagacaacagcagtgaagagtgaacacacgggaaaggagatagttgaccttatgtgtcacgccactttcactatgcgtctcctgtctcctgtgagagttcccaattataatatgattatcatggatgaagcacattttaccgatccagccagcatagcagccagagggtatatctcaacccgagtgggtatgggtgaagcagctgcgattttcatgacagccactccccccggatcggtggaggcctttccacagagcaatgcagttatccaagatgaggaaagagacattcctgaaagatcatggaactcaggctatgactggatcactgatttcccaggtaaaacagtctggtttgttccaagcatcaaatcaggaaatgacattgccaactgtttaagaaagaatgggaaacgggtggtccaattgagcagaaaaacttttgacactgagtaccagaaaacaaaaaataacgactgggactatgttgtcacaacagacatatccgaaatgggagcaaacttccgagccgacagggtaatagacccgaggcggtgcctgaaaccggtaatactaaaagatggcccagagcgtgtcattctagccggaccgatgccagtgactgtggctagcgccgcccagaggagaggaagaattggaaggaaccaaaataaggaaggcgatcagtatatttacatgggacagcctctaaacaatgatgaggaccacgcccattggacagaagcaaaaatgctccttgacaacataaacacaccagaagggattatcccagccctctttgagccggagagagaaaagagtgcagcaatagacggggaatacagactacggggtgaagcgaggaaaacgttcgtggagctcatgagaagaggagatctacctgtctggctatcctacaaagttgcctcagaaggcttccagtactccgacagaaggtggtgctttgatggggaaaggaacaaccaggtgttggaggagaacatggacgtggagatctggacaaaagaaggagaaagaaagaaactacgaccccgctggctggatgccagaacatactctgacccactggctctgcgcgaattcaaagagttcgcagcaggaagaagaagcgtctcaggtgacctaatattagaaatagggaaacttccacaacatttaacgcaaagggcccagaacgccttggacaatctggttatgttgcacaactctgaacaaggaggaaaagcctatagacacgccatggaagaactaccagacaccatagaaacgttaatgctcctagctttgatagctgtgctgactggtggagtgacgttgttcttcctatcaggaaggggtctaggaaaaacatccattggcctactctgcgtgattgcctcaagtgcactgttatggatggccagtgtggaaccccattggatagcggcctctatcatactggagttctttctgatggtgttgcttattccagagccggacagacagcgcactccacaagacaaccagctagcatacgtggtgataggtctgttattcatgatattgacagtggcagccaatgagatgggattactggaaaccacaaagaaggacctggggattggtcatgcagctgctgaaaaccaccatcatgctgcaatgctggacgtagacctacatccagcttcagcctggactctctatgcagtggccacaacaattatcactcccatgatgagacacacaattgaaaacacaacggcaaatatttccctgacagctattgcaaaccaggcagctatattgatgggacttgacaagggatggccaatatcaaagatggacataggagttccacttctcgccttggggtgctattctcaggtgaacccgctgacgctgacagcggcggtattgatgctagtggctcattatgccataattggacccggactgcaagcaaaagctactagagaagctcaaaaaaggacagcagccggaataatgaaaaacccaactgtcgacgggatcgttgcaatagatttggaccctgtggtttacgatgcaaaatttgaaaaacagctaggccaaataatgttgttgatactttgcacatcacagatcctcctgatgcggaccacatgggccttgtgtgaatccatcacactagccactggacctctgactacgctttgggagggatctccaggaaaattctggaacaccacgatagcggtgtccatggcaaacatttttaggggaagttatctagcaggagcaggtctggccttttcattaatgaaatctctaggaggaggtaggagaggcacgggagcccaaggggaaacactgggagaaaaatggaaaagacagctaaaccaattgagcaagtcagaattcaacacttacaaaaggagtgggattatagaggtggatagatctgaagccaaagaggggttaaaaagaggagaaacgactaaacacgcagtgtcgagaggaacggccaaactgaggtggtttgtggagaggaaccttgtgaaaccagaagggaaagtcatagacctcggttgtggaagaggtggctggtcatattattgcgctgggctgaagaaagtcacagaagtgaaaggatacacgaaaggaggacctggacatgaggaaccaatcccaatggcaacctatggatggaacctagtaaagctatactccgggaaagatgtattctttacaccacctgagaaatgtgacaccctcttgtgtgatattggtgagtcctctccgaacccaactatagaagaaggaagaacgttacgtgttctaaagatggtggaaccatggctcagaggaaaccaattttgcataaaaattctaaatccctatatgccgagtgtggtagaaactttggagcaaatgcaaagaaaacatggaggaatgctagtgcgaaatccactctcaagaaactccactcatgaaatgtactgggtttcatgtggaacaggaaacattgtgtcagcagtaaacatgacatctagaatgctgctaaatcgattcacaatggctcacaggaagccaacatatgaaagagacgtggacttaggcgctggaacaagacatgtggcagtagaaccagaggtggccaacctagatatcattggccagaggatagagaatataaaaaatgaacacaaatcaacatggcattatgatgaggacaatccatacaaaacatgggcctatcatggatcatatgaggtcaagccatcaggatcagcctcatccatggtcaatggtgtggtgagactgctaaccaaaccatgggatgtcattcccatggtcacacaaatagccatgactgacaccacaccctttggacaacagagggtgtttaaagagaaagttgacacgcgtacaccaaaagcgaaacgaggcacagcacaaattatggaggtgacagccaggtggttatggggttttctctctagaaacaaaaaacccagaatctgcacaagagaggagttcacaagaaaagtcaggtcaaacgcagctattggagcagtgttcgttgatgaaaatcaatggaactcagcaaaagaggcagtggaagatgaacggttctgggaccttgtgcacagagagagggagcttcataaacaaggaaaatgtgccacgtgtgtctacaacatgatgggaaagagagagaaaaaattaggagagttcggaaaggcaaaaggaagtcgcgcaatatggtacatgtggttgggagcgcgctttttagagtttgaagcccttggtttcatgaatgaagatcactggttcagcagagagaattcactcagtggagtggaaggagaaggactccacaaacttggatacatactcagagacatatcaaagattccagggggaaatatgtatgcagatgacacagccggatgggacacaagaataacagaggatgatcttcagaatgaggccaaaatcactgacatcatggaacctgaacatgccctattggccacgtcaatctttaagctaacctaccaaaacaaggtagtaagggtgcagagaccagcgaaaaatggaaccgtgatggatgtcatatccagacgtgaccagagaggaagtggacaggttggaacctatggcttaaacaccttcaccaacatggaggcccaactaataagacaaatggagtctgagggaatcttttcacccagcgaattggaaaccccaaatctagccgaaagagtcctcgactggttgaaaaaacatggcaccgagaggctgaaaagaatggcaatcagtggagatgactgtgtggtgaaaccaatcgatgacagatttgcaacagccttaacagctttgaatgacatgggaaaggtaagaaaagacataccgcaatgggaaccttcaaaaggatggaatgattggcaacaagtgcctttctgttcacaccatttccaccagctgattatgaaggatgggagggagatagtggtgccatgccgcaaccaagatgaacttgtaggtagggccagagtatcacaaggcgccggatggagcttgagagaaactgcatgcctaggcaagtcatatgcacaaatgtggcagctgatgtacttccacaggagagacttgagattagcggctaatgctatctgttcagccgttccagttgattgggtcccaaccagccgcaccacctggtcgatccatgcccaccatcaatggatgacaacagaagacatgttgtcagtgtggaatagggtttggatagaggaaaacccatggatggaggacaagactcatgtgtccagttgggaagacgttccatacctaggaaaaagggaagatcaatggtgtggttccctaataggcttaacagcacgagccacctgggccaccaacatacaagtggccataaaccaagtgagaaggctcattgggaatgagaattatctagacttcatgacatcaatgaagagattcaaaaacgagagtgatcccgaaggggcactctggtaagccaactcattcacaaaataaaggaaaataaaaaatcaaacaaggcaagaagtcaggccggattaagccatagcacggtaagagctatgctgcctgtgagccccgtccaaggacgtaaaatgaagtcaggccgaaagccacggttcgagcaagccgtgctgcctgtagctccatcgtggggatgtaaaaacccgggaggctgcaaaccatggaagctgtacgcatggggtagcagactagtggttagaggagacccctcccaagacacaacgcagcagcggggcccaacaccaggggaagctgtaccctggtggtaaggactagaggttagaggagaccccccgcacaacaacaaacagcatattgacgctgggagagaccagagatcctgctgtctctacagcatcattccaggcacagaacgccaaaaaatggaatggtgctgttgaatcaacaggttct DEN-2MNNQRKKAKNTPFNMLKRERNRVSTVQQLTKRFSLGMLQGRGPLKLFMAL 17 (NC_001474.2)VAFLRFLTIPPTAGILKRWGTIKKSKAINVLRGFRKEIGRMLNILNRRRRSAGMIIMLIPTVMAFHLTTRNGEPHMIVSRQEKGKSLLFKTEDGVNMCTLMAMDLGELCEDTITYKCPLLRQNEPEDIDCWCNSTSTWVTYGTCTTMGEHRREKRSVALVPHVGMGLETRTETWMSSEGAWKHVQRIETWILRHPGFTMMAAILAYTIGTTHFQRALIFILLTAVTPSMTMRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRSTSLSVTLVLVGIVTLYLGVMVQADSGCVVSWKNKELKCGSGIFITDNVHTWTEQYKFQPESPSKLASAIQKAHEEGICGIRSVTRLENLMWKQITPELNHILSENEVKLTIMTGDIKGIMQAGKRSLRPQPTELKYSWKTWGKAKMLSTESHNQTFLIDGPETAECPNTNRAWNSLEVEDYGFGVFTTNIWLKLKEKQDVFCDSKLMSAAIKDNRAVHADMGYWIESALNDTWKIEKASFIEVKNCHWPKSHTLWSNGVLESEMIIPKNLAGPVSQHNYRPGYHTQITGPWHLGKLEMDFDFCDGTTVVVTEDCGNRGPSLRTTTASGKLITEWCCRSCTLPPLRYRGEDGCWYGMEIRPLKEKEENLVNSLVTAGHGQVDNFSLGVLGMALFLEEMLRTRVGTKHAILLVAVSFVTLITGNMSFRDLGRVMVMVGATMTDDIGMGVTYLALLAAFKVRPTFAAGLLLRKLTSKELMMTTIGIVLLSQSTIPETILELTDALALGMMVLKMVRNMEKYQLAVTIMAILCVPILQNAWKVSCTILAVVSVSPLLLTSSQQKTDWIPLALTIKGLNPTAIFLTTLSRTSKKRSWPLNEAIMAVGMVSILASSLLKNDIPMTGPLVAGGLLTVCYVLTGRSADLELERAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLTILIRTGLLVISGLFPVSIPITAAAWYLWEVKKQRAGVLWDVPSPPPMGKAELEDGAYRIKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKKDLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIGAVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIEDNPEIEDDIFRKRRLTIMDLHPGAGKTKRYLPAIVREAIKRGLRTLILAPTRVVAAEMEEALRGLPIRYQTPAIRAEHTGREIVDLMCHATFTMRLLSPVRVPNYNLIIMDEAHFTDPASIAARGYISTRVEMGEAAGIFMTATPPGSRDPFPQSNAPIIDEEREIPERSWNSGHEWVTDFKGKTVWFVPSIKAGNDIAACLRKNGKKVIQLSRKTFDSEYVKTRTNDWDFVVTTDISEMGANFKAERVIDPRRCMKPVILTDGEERVILAGPMPVTHSSAAQRRGRIGRNPKNENDQYIYMGEPLENDEDCAHWKEAKMLLDNINTPEGIIPSMFEPEREKVDAIDGEYRLRGEARKTFVDLMRRGDLPVWLAYRVAAEGINYADRRWCFDGVKNNQILEENVEVEIWTKEGERKKLKPRWLDARIYSDPLALKEFKEFAAGRKSLTLNLITEMGRLPTFMTQKARDALDNLAVLHTAEAGGRAYNHALSELPETLETLLLLTLLATVTGGIFLFLMSGRGIGKMTLGMCCIITASILLWYAQIQPHWIAASIILEFFLIVLLIPEPEKQRTPQDNQLTYVVIAILTVVAATMANEMGFLEKTKKDLGLGSIATQQPESNILDIDLRPASAWTLYAVATTFVTPMLRHSIENSSVNVSLTAIANQATVLMGLGKGWPLSKMDIGVPLLAIGCYSQVNPITLTAALFLLVAHYAIIGPGLQAKATREAQKRAAAGIMKNPTVDGITVIDLDPIPYDPKFEKQLGQVMLLVLCVTQVLMMRTTWALCEALTLATGPISTLWEGNPGRFWNTTIAVSMANIFRGSYLAGAGLLFSIMKNTTNTRRGTGNIGETLGEKWKSRLNALGKSEFQIYKKSGIQEVDRTLAKEGIKRGETDHHAVSRGSAKLRWFVERNMVTPEGKVVDLGCGRGGWSYYCGGLKNVREVKGLTKGGPGHEEPIPMSTYGWNLVRLQSGVDVFFIPPEKCDTLLCDIGESSPNPTVEAGRTLRVLNLVENWLNNNTQFCIKVLNPYMPSVIEKMEALQRKYGGALVRNPLSRNSTHEMYWVSNASGNIVSSVNMISRMLINRFTMRYKKATYEPDVDLGSGTRNIGIESEIPNLDIIGKRIEKIKQEHETSWHYDQDHPYKTWAYHGSYETKQTGSASSMVNGVVRLLTKPWDVVPMVTQMAMTDTTPFGQQRVFKEKVDTRTQEPKEGTKKLMKITAEWLWKELGKKKTPRMCTREEFTRKVRSNAALGAIFTDENKWKSAREAVEDSRFWELVDKERNLHLEGKCETCVYNMMGKREKKLGEFGKAKGSRAIWYMWLGARFLEFEALGFLNEDHWFSRENSLSGVEGEGLHKLGYILRDVSKKEGGAMYADDTAGWDTRITLEDLKNEEMVTNHMEGEHKKLAEAIFKLTYQNKVVRVQRPTPRGTVMDIISRRDQRGSGQVGTYGLNTFTNMEAQLIRQMEGEGVFKSIQHLTITEEIAVQNWLARVGRERLSRMAISGDDCVVKPLDDRFASALTALNDMGKIRKDIQQWEPSRGWNDWTQVPFCSHHFHELIMKDGRVLVVPCRNQDELIGRARISQGAGWSLRETACLGKSYAQMWSLMYFHRRDLRLAANAICSAVPSHWVPTSRTTWSIHAKHEWMTTEDMLTVWNRVWIQENPWMEDKTPVESWEEIPYLGKREDQWCGSLIGLTSRATWAKNIQAAINQVRSLIGNEEYTDYMPSMKRFRREEEEAG VLW DEN-2agttgttagtctacgtggaccgacaaagacagattctttgagggagctaa 18 (NC_001474.2)gctcaacgtagttctaacagttttttaattagagagcagatctctgatgaataaccaacggaaaaaggcgaaaaacacgcctttcaatatgctgaaacgcgagagaaaccgcgtgtcgactgtgcaacagctgacaaagagattctcacttggaatgctgcagggacgaggaccattaaaactgttcatggccctggtggcgttccttcgtttcctaacaatcccaccaacagcagggatattgaagagatggggaacaattaaaaaatcaaaagctattaatgttttgagagggttcaggaaagagattggaaggatgctgaacatcttgaataggagacgcagatctgcaggcatgatcattatgctgattccaacagtgatggcgttccatttaaccacacgtaacggagaaccacacatgatcgtcagcagacaagagaaagggaaaagtcttctgtttaaaacagaggatggcgtgaacatgtgtaccctcatggccatggaccttggtgaattgtgtgaagacacaatcacgtacaagtgtccccttctcaggcagaatgagccagaagacatagactgttggtgcaactctacgtccacgtgggtaacttatgggacgtgtaccaccatgggagaacatagaagagaaaaaagatcagtggcactcgttccacatgtgggaatgggactggagacacgaactgaaacatggatgtcatcagaaggggcctggaaacatgtccagagaattgaaacttggatcttgagacatccaggcttcaccatgatggcagcaatcctggcatacaccataggaacgacacatttccaaagagccctgattttcatcttactgacagctgtcactccttcaatgacaatgcgttgcataggaatgtcaaatagagactttgtggaaggggtttcaggaggaagctgggttgacatagtcttagaacatggaagctgtgtgacgacgatggcaaaaaacaaaccaacattggattttgaactgataaaaacagaagccaaacagcctgccaccctaaggaagtactgtatagaggcaaagctaaccaacacaacaacagaatctcgctgcccaacacaaggggaacccagcctaaatgaagagcaggacaaaaggttcgtctgcaaacactccatggtagacagaggatggggaaatggatgtggactatttggaaagggaggcattgtgacctgtgctatgttcagatgcaaaaagaacatggaaggaaaagttgtgcaaccagaaaacttggaatacaccattgtgataacacctcactcaggggaagagcatgcagtcggaaatgacacaggaaaacatggcaaggaaatcaaaataacaccacagagttccatcacagaagcagaattgacaggttatggcactgtcacaatggagtgctctccaagaacgggcctcgacttcaatgagatggtgttgctgcagatggaaaataaagcttggctggtgcacaggcaatggttcctagacctgccgttaccatggttgcccggagcggacacacaagggtcaaattggatacagaaagagacattggtcactttcaaaaatccccatgcgaagaaacaggatgttgttgttttaggatcccaagaaggggccatgcacacagcacttacaggggccacagaaatccaaatgtcatcaggaaacttactcttcacaggacatctcaagtgcaggctgagaatggacaagctacagctcaaaggaatgtcatactctatgtgcacaggaaagtttaaagttgtgaaggaaatagcagaaacacaacatggaacaatagttatcagagtgcaatatgaaggggacggctctccatgcaagatcccttttgagataatggatttggaaaaaagacatgtcttaggtcgcctgattacagtcaacccaattgtgacagaaaaagatagcccagtcaacatagaagcagaacctccattcggagacagctacatcatcataggagtagagccgggacaactgaagctcaactggtttaagaaaggaagttctatcggccaaatgtttgagacaacaatgaggggggcgaagagaatggccattttaggtgacacagcctgggattttggatccttgggaggagtgtttacatctataggaaaggctctccaccaagtctttggagcaatctatggagctgccttcagtggggtttcatggactatgaaaatcctcataggagtcattatcacatggataggaatgaattcacgcagcacctcactgtctgtgacactagtattggtgggaattgtgacactgtatttgggagtcatggtgcaggccgatagtggttgcgttgtgagctggaaaaacaaagaactgaaatgtggcagtgggattttcatcacagacaacgtgcacacatggacagaacaatacaagttccaaccagaatccccttcaaaactagcttcagctatccagaaagcccatgaagagggcatttgtggaatccgctcagtaacaagactggagaatctgatgtggaaacaaataacaccagaattgaatcacattctatcagaaaatgaggtgaagttaactattatgacaggagacatcaaaggaatcatgcaggcaggaaaacgatctctgcggcctcagcccactgagctgaagtattcatggaaaacatggggcaaagcaaaaatgctctctacagagtctcataaccagacctttctcattgatggccccgaaacagcagaatgccccaacacaaatagagcttggaattcgttggaagttgaagactatggctttggagtattcaccaccaatatatggctaaaattgaaagaaaaacaggatgtattctgcgactcaaaactcatgtcagcggccataaaagacaacagagccgtccatgccgatatgggttattggatagaaagtgcactcaatgacacatggaagatagagaaagcctctttcattgaagttaaaaactgccactggccaaaatcacacaccctctggagcaatggagtgctagaaagtgagatgataattccaaagaatctcgctggaccagtgtctcaacacaactatagaccaggctaccatacacaaataacaggaccatggcatctaggtaagcttgagatggactttgatttctgtgatggaacaacagtggtagtgactgaggactgcggaaatagaggaccctctttgagaacaaccactgcctctggaaaactcataacagaatggtgctgccgatcttgcacattaccaccgctaagatacagaggtgaggatgggtgctggtacgggatggaaatcagaccattgaaggagaaagaagagaatttggtcaactccttggtcacagctggacatgggcaggtcgacaacttttcactaggagtcttgggaatggcattgttcctggaggaaatgcttaggacccgagtaggaacgaaacatgcaatactactagttgcagtttcttttgtgacattgatcacagggaacatgtcctttagagacctgggaagagtgatggttatggtaggcgccactatgacggatgacataggtatgggcgtgacttatcttgccctactagcagccttcaaagtcagaccaacttttgcagctggactactcttgagaaagctgacctccaaggaattgatgatgactactataggaattgtactcctctcccagagcaccataccagagaccattcttgagttgactgatgcgttagccttaggcatgatggtcctcaaaatggtgagaaatatggaaaagtatcaattggcagtgactatcatggctatcttgtgcgtcccaaacgcagtgatattacaaaacgcatggaaagtgagttgcacaatattggcagtggtgtccgtttccccactgctcttaacatcctcacagcaaaaaacagattggataccattagcattgacgatcaaaggtctcaatccaacagctatttttctaacaaccctctcaagaaccagcaagaaaaggagctggccattaaatgaggctatcatggcagtcgggatggtgagcattttagccagttctctcctaaaaaatgatattcccatgacaggaccattagtggctggagggctcctcactgtgtgctacgtgctcactggacgatcggccgatttggaactggagagagcagccgatgtcaaatgggaagaccaggcagagatatcaggaagcagtccaatcctgtcaataacaatatcagaagatggtagcatgtcgataaaaaatgaagaggaagaacaaacactgaccatactcattagaacaggattgctggtgatctcaggactttttcctgtatcaataccaatcacggcagcagcatggtacctgtgggaagtgaagaaacaacgggccggagtattgtgggatgttccttcacccccacccatgggaaaggctgaactggaagatggagcctatagaattaagcaaaaagggattcttggatattcccagatcggagccggagtttacaaagaaggaacattccatacaatgtggcatgtcacacgtggcgctgttctaatgcataaaggaaagaggattgaaccatcatgggcggacgtcaagaaagacctaatatcatatggaggaggctggaagttagaaggagaatggaaggaaggagaagaagtccaggtattggcactggagcctggaaaaaatccaagagccgtccaaacgaaacctggtcttttcaaaaccaacgccggaacaataggtgctgtatctctggacttttctcctggaacgtcaggatctccaattatcgacaaaaaaggaaaagttgtgggtctttatggtaatggtgttgttacaaggagtggagcatatgtgagtgctatagcccagactgaaaaaagcattgaagacaacccagagatcgaagatgacattttccgaaagagaagactgaccatcatggacctccacccaggagcgggaaagacgaagagataccttccggccatagtcagagaagctataaaacggggtttgagaacattaatcttggcccccactagagttgtggcagctgaaatggaggaagcccttagaggacttccaataagataccagaccccagccatcagagctgagcacaccgggcgggagattgtggacctaatgtgtcatgccacatttaccatgaggctgctatcaccagttagagtgccaaactacaacctgattatcatggacgaagcccatttcacagacccagcaagtatagcagctagaggatacatctcaactcgagtggagatgggtgaggcagctgggatttttatgacagccactcccccgggaagcagagacccatttcctcagagcaatgcaccaatcatagatgaagaaagagaaatccctgaacgttcgtggaattccggacatgaatgggtcacggattttaaagggaagactgtttggttcgttccaagtataaaagcaggaaatgatatagcagcttgcctgaggaaaaatggaaagaaagtgatacaactcagtaggaagacctttgattctgagtatgtcaagactagaaccaatgattgggacttcgtggttacaactgacatttcagaaatgggtgccaatttcaaggctgagagggttatagaccccagacgctgcatgaaaccagtcatactaacagatggtgaagagcgggtgattctggcaggacctatgccagtgacccactctagtgcagcacaaagaagagggagaataggaagaaatccaaaaaatgagaatgaccagtacatatacatgggggaacctctggaaaatgatgaagactgtgcacactggaaagaagctaaaatgctcctagataacatcaacacgccagaaggaatcattcctagcatgttcgaaccagagcgtgaaaaggtggatgccattgatggcgaataccgcttgagaggagaagcaaggaaaacctttgtagacttaatgagaagaggagacctaccagtctggttggcctacagagtggcagctgaaggcatcaactacgcagacagaaggtggtgttttgatggagtcaagaacaaccaaatcctagaagaaaacgtggaagttgaaatctggacaaaagaaggggaaaggaagaaattgaaacccagatggttggatgctaggatctattctgacccactggcgctaaaagaatttaaggaatttgcagccggaagaaagtctctgaccctgaacctaatcacagaaatgggtaggctcccaaccttcatgactcagaaggcaagagacgcactggacaacttagcagtgctgcacacggctgaggcaggtggaagggcgtacaaccatgctctcagtgaactgccggagaccctggagacattgcttttactgacacttctggctacagtcacgggagggatctttttattcttgatgagcggaaggggcatagggaagatgaccctgggaatgtgctgcataatcacggctagcatcctcctatggtacgcacaaatacagccacactggatagcagcttcaataatactggagttttttctcatagttttgcttattccagaacctgaaaaacagagaacaccccaagacaaccaactgacctacgttgtcatagccatcctcacagtggtggccgcaaccatggcaaacgagatgggtttcctagaaaaaacgaagaaagatctcggattgggaagcattgcaacccagcaacccgagagcaacatcctggacatagatctacgtcctgcatcagcatggacgctgtatgccgtggccacaacatttgttacaccaatgttgagacatagcattgaaaattcctcagtgaatgtgtccctaacagctatagccaaccaagccacagtgttaatgggtctcgggaaaggatggccattgtcaaagatggacatcggagttccccttctcgccattggatgctactcacaagtcaaccccataactctcacagcagctcttttcttattggtagcacattatgccatcatagggccaggactccaagcaaaagcaaccagagaagctcagaaaagagcagcggcgggcatcatgaaaaacccaactgtcgatggaataacagtgattgacctagatccaataccttatgatccaaagtttgaaaagcagttgggacaagtaatgctcctagtcctctgcgtgactcaagtattgatgatgaggactacatgggctctgtgtgaggctttaaccttagctaccgggcccatctccacattgtgggaaggaaatccagggaggttttggaacactaccattgcggtgtcaatggctaacatttttagagggagttacttggccggagctggacttctcttttctattatgaagaacacaaccaacacaagaaggggaactggcaacataggagagacgcttggagagaaatggaaaagccgattgaacgcattgggaaaaagtgaattccagatctacaagaaaagtggaatccaggaagtggatagaaccttagcaaaagaaggcattaaaagaggagaaacggaccatcacgctgtgtcgcgaggctcagcaaaactgagatggttcgttgagagaaacatggtcacaccagaagggaaagtagtggacctcggttgtggcagaggaggctggtcatactattgtggaggactaaagaatgtaagagaagtcaaaggcctaacaaaaggaggaccaggacacgaagaacccatccccatgtcaacatatgggtggaatctagtgcgtcttcaaagtggagttgacgttttcttcatcccgccagaaaagtgtgacacattattgtgtgacataggggagtcatcaccaaatcccacagtggaagcaggacgaacactcagagtccttaacttagtagaaaattggttgaacaacaacactcaattttgcataaaggttctcaacccatatatgccctcagtcatagaaaaaatggaagcactacaaaggaaatatggaggagccttagtgaggaatccactctcacgaaactccacacatgagatgtactgggtatccaatgcttccgggaacatagtgtcatcagtgaacatgatttcaaggatgttgatcaacagatttacaatgagatacaagaaagccacttacgagccggatgttgacctcggaagcggaacccgtaacatcgggattgaaagtgagataccaaacctagatataattgggaaaagaatagaaaaaataaagcaagagcatgaaacatcatggcactatgaccaagaccacccatacaaaacgtgggcataccatggtagctatgaaacaaaacagactggatcagcatcatccatggtcaacggagtggtcaggctgctgacaaaaccttgggacgtcgtccccatggtgacacagatggcaatgacagacacgactccatttggacaacagcgcgtttttaaagagaaagtggacacgagaacccaagaaccgaaagaaggcacgaagaaactaatgaaaataacagcagagtggctttggaaagaattagggaagaaaaagacacccaggatgtgcaccagagaagaattcacaagaaaggtgagaagcaatgcagccttgggggccatattcactgatgagaacaagtggaagtcggcacgtgaggctgttgaagatagtaggttttgggagctggttgacaaggaaaggaatctccatcttgaaggaaagtgtgaaacatgtgtgtacaacatgatgggaaaaagagagaagaagctaggggaattcggcaaggcaaaaggcagcagagccatatggtacatgtggcttggagcacgcttcttagagtttgaagccctaggattcttaaatgaagatcactggttctccagagagaactccctgagtggagtggaaggagaagggctgcacaagctaggttacattctaagagacgtgagcaagaaagagggaggagcaatgtatgccgatgacaccgcaggatgggatacaagaatcacactagaagacctaaaaaatgaagaaatggtaacaaaccacatggaaggagaacacaagaaactagccgaggccattttcaaactaacgtaccaaaacaaggtggtgcgtgtgcaaagaccaacaccaagaggcacagtaatggacatcatatcgagaagagaccaaagaggtagtggacaagttggcacctatggactcaatactttcaccaatatggaagcccaactaatcagacagatggagggagaaggagtctttaaaagcattcagcacctaacaatcacagaagaaatcgctgtgcaaaactggttagcaagagtggggcgcgaaaggttatcaagaatggccatcagtggagatgattgtgttgtgaaacctttagatgacaggttcgcaagcgctttaacagctctaaatgacatgggaaagattaggaaagacatacaacaatgggaaccttcaagaggatggaatgattggacacaagtgcccttctgttcacaccatttccatgagttaatcatgaaagacggtcgcgtactcgttgttccatgtagaaaccaagatgaactgattggcagagcccgaatctcccaaggagcagggtggtctttgcgggagacggcctgtttggggaagtcttacgcccaaatgtggagcttgatgtacttccacagacgcgacctcaggctggcggcaaatgctatttgctcggcagtaccatcacattgggttccaacaagtcgaacaacctggtccatacatgctaaacatgaatggatgacaacggaagacatgctgacagtctggaacagggtgtggattcaagaaaacccatggatggaagacaaaactccagtggaatcatgggaggaaatcccatacttggggaaaagagaagaccaatggtgcggctcattgattgggttaacaagcagggccacctgggcaaagaacatccaagcagcaataaatcaagttagatcccttataggcaatgaagaatacacagattacatgccatccatgaaaagattcagaagagaagaggaagaagcaggagttctgtggtagaaagcaaaactaacatgaaacaaggctagaagtcaggtcggattaagccatagtacggaaaaaactatgctacctgtgagccccgtccaaggacgttaaaagaagtcaggccatcataaatgccatagcttgagtaaactatgcagcctgtagctccacctgagaaggtgtaaaaaatccgggaggccacaaaccatggaagctgtacgcatggcgtagtggactagcggttagaggagacccctcccttacaaatcgcagcaacaatgggggcccaaggcgagatgaagctgtagtctcgctggaaggactagaggttagaggagacccccccgaaacaaaaaacagcatattgacgctgggaaagaccagagatcctgctgtctcctcagcatcattccaggcacagaacgccagaaaatggaatggtgctgttgaatcaacaggttct DEN-3MNNQRKKTGKPSINMLKRVRNRVSTGSQLAKRFSKGLLNGQGPMKLVMAF 19 (NC_001475.2)IAFLRFLAIPPTAGVLARWGTFKKSGAIKVLKGFKKEISNMLSIINQRKKTSLCLMMILPAALAFHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTLIAMDLGEMCDDTVTYKCPHITEVEPEDIDCWCNLTSTWVTYGTCNQAGEHRRDKRSVALAPHVGMGLDTRTQTWMSAEGAWRQVEKVETWALRHPGFTILALFLAHYIGTSLTQKVVIFILLMLVTPSMTMRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQGEAVLPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQCLEPIEGKVVQYENLKYTVIITVHTGDQHQVGNETQGVTAEITPQASTTEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWASGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYAMCTNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDNALKINWYKKGSSIGKMFEATERGARRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSAYTALFSGVSWVMKIGIGVLLTWIGLNSKNTSMSFSCIAIGIITLYLGAVVQADMGCVINWKGKELKCGSGIFVTNEVHTWTEQYKFQADSPKRLATAIAGAWENGVCGIRSTTRMENLLWKQIANELNYILWENNIKLTVVVGDTLGVLEQGKRTLTPQPMELKYSWKTWGKAKIVTAETQNSSFIIDGPNTPECPSASRAWNVWEVEDYGFGVFTTNIWLKLREVYTQLCDHRLMSAAVKDERAVHADMGYWIESQKNGSWKLEKASLIEVKTCTWPKSHTLWTNGVLESDMIIPKSLAGPISQHNYRPGYHTQTAGPWHLGKLELDFNYCEGTTVVITESCGTRGPSLRTTTVSGKLIHEWCCRSCTLPPLRYMGEDGCWYGMEIRPISEKEENMVKSLVSAGSGKVDNFTMGVLCLAILFEEVLRGKFGKKHMIAGVFFTFVLLLSGQITWRDMAHTLIMIGSNASDRMGMGVTYLALIATFKIQPFLALGFFLRKLTSRENLLLGVGLAMATTLQLPEDIEQMANGVALGLMALKLITQFETYQLWTALVSLTCSNTIFTLTVAWRTATLILAGVSLLPVCQSSSMRKTDWLPMTVAAMGVPPLPLFIFSLKDTLKRRSWPLNEGVMAVGLVSILASSLLRNDVPMAGPLVAGGLLIACYVITGTSADLTVEKAPDVTWEEEAEQTGVSHNLMITVDDDGTMRIKDDETENILTVLLKTALLIVSGIFPYSIPATLLVWHTWQKQTQRSGVLWDVPSPPETQKAELEEGVYRIKQQGIFGKTQVGVGVQKEGVFHTMWHVTRGAVLTHNGKRLEPNWASVKKDLISYGGGWRLSAQWQKGEEVQVIAVEPGKNPKNFQTTPGTFQTTTGEIGAIALDFKPGTSGSPIINREGKVVGLYGNGVVTKNGGYVSGIAQTNAEPDGPTPELEEEMFKKRNLTIMDLHPGSGKTRKYLPAIVREAIKRRLRTLILAPTRVVAAEMEEALKGLPIRYQTTATKSEHTGREIVDLMCHATFTMRLLSPVRVPNYNLIIMDEAHFTDPASIAARGYISTRVGMGEAAAIFMTATPPGTADAFPQSNAPIQDEERDIPERSWNSGNEWITDFAGKTVWFVPSIKAGNDIANCLRKNGKKVIQLSRKTFDTEYQKTKLNDWDFVVTTDISEMGANFKADRVIDPRRCLKPVILTDGPERVILAGPMPVTAASAAQRRGRVGRNPQKENDQYIFTGQPLNNDEDHAHWTEAKMLLDNINTPEGIIPALFEPEREKSAAIDGEYRLKGESRKTFVELMRRGDLPVWLAHKVASEGIKYTDRKWCFDGQRNNQILEENMDVEIWTKEGEKKKLRPRWLDARTYSDPLALKEFKDFAAGRKSIALDLVTEIGRVPSHLAHRTRNALDNLVMLHTSEDGGRAYRHAVEELPETMETLLLLGLMILLTGGAMLFLISGKGIGKTSIGLICVIASSGMLWMAEVPLQWIASAIVLEFFMMVLLIPEPEKQRTPQDNQLAYVVIGILTLAATIAANEMGLLETTKRDLGMSKEPGVVSPTSYLDVDLHPASAWTLYAVATTVITPMLRHTIENSTANVSLAAIANQAVVLMGLDKGWPISKMDLGVPLLALGCYSQVNPLTLTAAVLLLITHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGIMTIDLDSVIFDSKFEKQLGQVMLLVLCAVQLLLMRTSWALCEALTLATGPITTLWEGSPGKFWNTTIAVSMANIFRGSYLAGAGLAFSIMKSVGTGKRGTGSQGETLGEKWKKKLNQLSRKEFDLYKKSGITEVDRTEAKEGLKRGETTHHAVSRGSAKLQWFVERNMVVPEGRVIDLGCGRGGWSYYCAGLKKVTEVRGYTKGGPGHEEPVPMSTYGWNIVKLMSGKDVFYLPPEKCDTLLCDIGESSPSPTVEESRTIRVLKMVEPWLKNNQFCIKVLNPYMPTVIEHLERLQRKHGGMLVRNPLSRNSTHEMYWISNGTGNIVSSVNMVSRLLLNRFTMTHRRPTIEKDVDLGAGTRHVNAEPETPNMDVIGERIKRIKEEHNSTWHYDDENPYKTWAYHGSYEVKATGSASSMINGVVKLLTKPWDVVPMVTQMAMTDTTPFGQQRVFKEKVDTRTPRPMPGTRKAMEITAEWLWRTLGRNKRPRLCTREEFTKKVRTNAAMGAVFTEENQWDSAKAAVEDEEFWKLVDRERELHKLGKCGSCVYNMMGKREKKLGEFGKAKGSRAIWYMWLGARYLEFEALGFLNEDHWFSRENSYSGVEGEG LHKLGYILRDISKIPGGAMYADDTAGWDTRITEDDLHNEEKIIQQMDPEHRQLANAIFKLTYQNKVVKVQRPTPTGTVMDIISRKDQRGSGQLGTYGLNTFTNMEAQLVRQMEGEGVLTKADLENPHLLEKKITQWLETKGVERLKRMAISGDDCVVKPIDDRFANALLALNDMGKVRKDIPQWQPSKGWHDWQQVPFCSHHFHELIMKDGRKLVVPCRPQDELIGRARISQGAGWSLRETACLGKAYAQMWSLMYFHRRDLRLASNAICSAVPVHWVPTSRTTWSIHAHHQWMTTEDMLTVWNRVWIEENPWMEDKTPVTTWENVPYLGKREDQWCGSLIGLTSRATWAQNIPTAIQQVRSLIGNEEFLDYMPSMKRFRKEEESEGAIW DEN-3agttgttagtctacgtggaccgacaagaacagtttcgactcggaagcttg 20 (NC_001475.2)cttaacgtagtgctgacagttttttattagagagcagatctctgatgaacaaccaacggaagaagacgggaaaaccgtctatcaatatgctgaaacgcgtgagaaaccgtgtgtcaactggatcacagttggcgaagagattctcaaaaggactgctgaacggccagggaccaatgaaattggttatggcgttcatagctttcctcagatttctagccattccaccaacagcaggagtcttggctagatggggaaccttcaagaagtcgggggccattaaggtcctgaaaggcttcaagaaggagatctcaaacatgctgagcataatcaaccaacggaaaaagacatcgctctgtctcatgatgatattgccagcagcacttgctttccacttgacttcacgagatggagagccgcgcatgattgtggggaagaatgaaagaggtaaatccctactttttaagacagcctctggaatcaacatgtgcacactcatagccatggatttgggagagatgtgtgatgacacggtcacttacaaatgcccccacattaccgaagtggaacctgaagacattgactgctggtgcaaccttacatcaacatgggtgacttatggaacgtgcaatcaagctggagagcatagacgcgacaagagatcagtggcgttagctccccatgtcggcatgggactggacacacgcacccaaacctggatgtcggctgaaggagcttggagacaagtcgagaaggtagagacatgggcccttaggcacccagggttcaccatactagccctatttctcgcccattacataggcacttccctgacccagaaggtggttattttcatattattaatgctggtcaccccatccatgacaatgagatgtgtgggagtaggaaacagagattttgtggaagggctatcaggagctacgtgggttgacgtggtgctcgagcacggggggtgtgtgactaccatggctaagaacaagcccacgctggatatagagcttcagaagaccgaggccacccaactggcgaccctaaggaagctatgcattgaggggaaaattaccaacataacaactgactcaagatgtcctacccaaggggaagcggttttgcctgaggagcaggaccagaactacgtgtgtaagcatacatacgtagacagaggttgggggaacggttgtggtttgtttggcaaaggaagcttggtaacatgtgcgaaatttcaatgcctggaaccaatagagggaaaagtggtgcaatatgagaacctcaaatacaccgtcatcattacagtgcacacaggagaccaacaccaggtgggaaatgaaacgcaaggagtcacggctgagataacacctcaggcatcaaccactgaagccatcttgcctgaatatggaacccttgggctagaatgctcaccacggacaggtttggatttcaatgaaatgatcttactaacaatgaagaacaaagcatggatggtacatagacaatggttctttgacctacctctaccatgggcatcaggagctacaacagaaacaccaacctggaacaggaaggagcttcttgtgacattcaaaaacgcacatgcgaaaaaacaagaagtagttgtccttggatcgcaagagggagcaatgcataccgcactgacaggagctacagaaatccaaaactcaggaggcacaagcattttcgcggggcacttaaaatgtagacttaagatggacaaattggaactcaaggggatgagctatgcaatgtgcacgaatacctttgtgttgaagaaagaagtctcagaaacgcagcacgggacaatactcattaaggttgagtacaaaggggaagatgcaccttgcaagattcccttttccacagaggatggacaagggaaagctcataatggcagactgatcacagccaaccctgtggtgactaagaaggaggagcctgtcaatattgaggctgaacctccttttggggaaagcaatatagtaattggaattggagacaacgccttgaaaatcaactggtacaagaaggggagctcgattgggaagatgttcgaggccactgaaaggggtgcaaggcgcatggccatcttgggagacacagcttgggactttggatcagtgggtggtgttctgaactcattaggcaaaatggtgcaccaaatatttggaagtgcttatacagccctgttcagtggagtctcttgggtgatgaaaattggaataggtgtcctcttgacttggatagggttgaattcaaaaaacacatccatgtcattttcatgcattgcgataggaatcattacactctatctgggagctgtggtacaagctgacatggggtgtgtcataaactggaagggcaaagaactcaaatgtggaagcggaattttcgtcaccaatgaggtccatacctggacagagcaatacaaattccaagcagactccccaaaaagattggcaacagccattgcaggcgcctgggagaatggagtgtgtggaattaggtcaacaaccagaatggagaatctcttgtggaagcaaatagccaatgaactgaactacatattatgggaaaacaatatcaaattaacggtagttgtgggcgatacacttggggtcttagagcaagggaaaagaacactaacaccacaacccatggagctaaaatactcatggaaaacgtggggaaaggcaaaaatagtgacagctgaaacacaaaattcctctttcataatagacgggccaaacacaccggagtgtccaagtgcctcaagagcatggaatgtgtgggaggtggaagattacgggttcggagtcttcacaaccaacatatggctgaaactccgagaggtctacacccaactatgtgaccataggctaatgtcggcagctgtcaaggatgagagggccgtgcatgccgacatgggctactggatagaaagccaaaagaatggaagttggaagctagaaaaagcatccctcatagaggtaaaaacctgcacatggccaaaatcacacactctctggactaatggtgtgctagagagtgacatgatcatcccaaagagtctagctggtcctatctcacaacacaactacaggcccgggtaccacacccaaacggcaggaccctggcacttaggaaaattggagctggacttcaactactgtgaaggaacaacagttgtcatcacagaaagctgtgggacaagaggcccatcattgagaacaacaacagtgtcagggaagttgatacacgaatggtgttgccgctcgtgcacacttccccccctgcgatacatgggagaagacggctgctggtatggcatggaaatcagacccatcagtgagaaagaagagaacatggtaaagtctttagtctcagcgggaagtggaaaggtggacaacttcacaatgggtgtcttgtgtttggcaatcctctttgaagaggtgttgagaggaaaatttgggaagaaacacatgattgcaggggttttctttacgtttgtgctccttctctcagggcaaataacatggagagacatggcgcacacactaataatgatcgggtccaacgcctctgacaggatgggaatgggcgtcacctacctagctctaattgcaacatttaaaatccagccattcttggctttgggatttttcctaagaaagctgacatctagagaaaatttattgttaggagttgggttggccatggcaacaacgttacaactgccagaggacattgaacaaatggcaaatggagtcgctctggggctcatggctcttaaactgataacacaatttgaaacataccaattgtggacggcattagtctccttaacgtgttcaaacacaatttttacgttgactgttgcctggagaacagccactctgattttggccggagtttcgcttttaccagtgtgccagtcttcaagcatgaggaaaacagattggctcccaatgacagtggcagctatgggagttccaccccttccactttttatttttagcttgaaagacacactcaaaaggagaagctggccactgaatgaaggggtgatggctgttgggcttgtgagcattctggccagttctctccttagaaatgatgtgcccatggctggaccattagtggccgggggcttgctgatagcgtgctacgtcataactggcacgtcagcggacctcactgtagaaaaagccccagatgtaacatgggaggaagaggctgagcagacaggagtgtcccacaacttaatgatcacagttgatgatgatggaacaatgagaataaaagatgatgagactgagaacatcctaacagtgcttttaaaaacagcattactaatagtatcaggcatttttccatactccatacccgcaacattgttggtctggcacacttggcaaaaacaaacccaaagatccggcgttttatgggacgtacccagccccccagagacacagaaagcagaactggaagaaggggtttataggatcaaacagcaaggaatttttgggaaaacccaagtaggggttggagtacagaaagaaggagtcttccacaccatgtggcacgtcacaagaggggcagtgttgacacataatgggaaaagactggaaccaaactgggctagtgtgaaaaaagatctgatttcatatggaggaggatggagactgagcgcacaatggcaaaagggggaggaggtgcaggttattgccgtagagccagggaagaacccaaagaactttcaaaccacgccaggcactttccagactactacaggggaaataggagcaattgcactggatttcaagcctggaacttcaggatctcctatcataaatagagagggaaaggtagtgggactgtatggcaatggagtggttacaaagaatggtggctatgtcagcggaatagcgcaaacaaatgcagaaccagatggaccgacaccagagttggaagaagagatgttcaaaaagcgaaacctgaccataatggatcttcatcctgggtcaggaaagacacggaaataccttccagctattgtcagagaggcaatcaagagacgtttaagaaccttaattttggcaccgacaagggtggttgcagctgagatggaagaagcattgaaagggctcccaataaggtaccaaacaacagcaacaaaatctgaacacacaggaagagagattgttgatctaatgtgccacgcaacgttcacaatgcgtttgctgtcaccagttagggttccaaattacaacttgataataatggatgaggcccatttcacagacccagccagtatagcggctagagggtacatatcaactcgtgttggaatgggagaggcagccgcaatcttcatgacagcaacaccccctggaacagctgatgcctttcctcagagcaacgctccaattcaagatgaagaaagggacataccagaacgctcatggaattcaggcaatgaatggattaccgacttcgctgggaaaacggtgtggtttgtccctagcattaaagccggaaatgacatagcaaactgcttgcgaaaaaacgggaaaaaagtcattcaacttagtaggaagacttttgacacagaatatcagaagactaaactgaatgattgggactttgtggtgacaactgacatttcagaaatgggggccaatttcaaagcagatagagtgatcgacccaagaagatgtctcaaaccagtgatcttgacagatggaccagagcgggtgatcctggccggaccaatgccagtcaccgcggcgagtgctgcgcaaaggagagggagagttggcaggaacccacaaaaagagaatgaccagtacatattcacgggccagcctctcaacaatgatgaagaccatgctcactggacagaagcaaaaatgctgctggacaacatcaacacaccagaagggattataccagctctctttgaaccagaaagggagaagtcagccgccatagacggtgagtatcgcctgaagggtgagtccaggaagactttcgtggaactcatgaggaggggtgaccttccagtttggttagcccataaagtagcatcagaaggaatcaaatacacagatagaaaatggtgctttgatgggcaacgcaataatcaaattttagaggagaacatggatgtggaaatttggacaaaggaaggagaaaagaaaaaattgagacctaggtggcttgatgcccgcacttattcagatccattggcactcaaggaattcaaggactttgcggctggcagaaagtcaatcgcccttgatcttgtgacagaaataggaagagtgccttcacatctagcccacagaacaagaaacgctctggacaatctggtgatgctgcatacgtcagaagatggcggtagggcttacaggcatgcggtggaggaactaccagaaacaatggaaacactcctactcttgggactaatgatcttgttgacaggtggagcaatgcttttcttgatatcaggtaaagggattggaaagacttcaataggactcatttgtgtaatcgcttccagcggcatgttgtggatggccgaagttccactccaatggatcgcgtcggctatagtcctggagttttttatgatggtgttgctcataccagaaccagaaaagcagagaaccccccaagacaaccaactcgcatatgtcgtgataggcatacttacattggctgcaacaatagcagccaatgaaatgggactgctggaaaccacaaagagagacttaggaatgtctaaggagccaggtgttgtttctccaaccagctatttggatgtggacttgcacccagcatcagcctggacattgtacgccgtggccactacagtaataacaccaatgttaagacataccatagagaattctacagcaaatgtgtccctggcagctatagccaaccaggcagtggtcctgatgggtttggacaaaggatggccaatatcaaaaatggacttaggcgtgccactactggcactgggttgctattcacaagtgaacccactgactctaactgcggcagtacttttgctaatcacacattatgctatcataggtccaggattgcaagcaaaagccacccgtgaagctcagaaaaggacagctgctggaataatgaagaatccaacagtggatgggataatgacaatagacctagattctgtaatatttgattcaaaatttgaaaaacaactgggacaggttatgctcctggttttgtgcgcagtccaactcttgctaatgagaacatcatgggccttgtgtgaagctttaactctagctacaggaccaataacaacactctgggaaggatcacctggtaagttctggaacaccacgatagctgtttccatggcgaacatttttagagggagctatttagcaggagctgggcttgctttttctattatgaaatcagttggaacaggaaaaagaggaacaggctcacaaggtgaaactttaggagaaaaatggaaaaagaaattaaatcaattatcccggaaagagtttgacctttacaagaaatctggaatcactgaagtggatagaacagaagccaaagaagggttgaaaagaggagagacaacacatcatgccgtgtcccgaggtagcgcaaaacttcaatggtttgtggaaagaaacatggtcgttcccgaaggaagagtcatagacttgggctgtggaagaggaggctggtcatattactgtgcaggactgaaaaaagtcacagaagtgcgaggatacacaaaaggcggtccaggacacgaagaaccagtacctatgtctacatatggatggaacatagttaagttaatgagcggaaaggatgtgttctatctcccacctgaaaagtgtgataccctgttgtgtgacattggagaatcttcaccaagcccaacagtggaagagagcagaactataagagttttgaagatggttgaaccatggctaaaaaacaaccagttttgcattaaagttttgaacccttacatgccaactgtgattgagcacctagaaagactacaaaggaaacatggaggaatgcttgtgagaaatccactttcacgaaactccacgcacgaaatgtactggatatctaatggcacaggtaacattgtctcttcagtcaacatggtgtctagattgctactgaacaggttcacgatgacacacaggagacccaccatagagaaagatgtggatttaggagcaggaactcgacatgttaatgcggaaccagaaacacccaacatggatgtcattggggaaagaataaaaaggatcaaggaggagcataattcaacatggcactatgatgacgaaaacccctacaaaacgtgggcttaccatggatcctatgaagtcaaagccacaggctcagcctcctccatgataaatggagtcgtgaaactcctcaccaaaccatgggatgtggtgcccatggtgacacagatggcaatgacagacacaactccatttggccagcagagagtctttaaagagaaagtggacaccaggacgcccaggcccatgccagggacaagaaaggctatggagatcacagcggagtggctctggagaaccctgggaaggaacaaaagacccagattatgcacaagggaagagtttacaaaaaaggtcagaactaacgcagccatgggcgccgttttcacagaggagaaccaatgggacagtgcgaaagctgctgttgaggatgaagaattttggaaacttgtggacagagaacgtgaactccacaaattgggcaaatgtggaagctgcgtttataacatgatgggcaagagagagaaaaaacttggagagtttggcaaagcaaaaggcagtagagctatatggtacatgtggttgggagccaggtaccttgagttcgaagcccttggattcttaaatgaagaccactggttctcgcgtgaaaactcttacagtggagtagaaggagaaggactgcacaagctaggctacatattaagggacatttccaagatacccggaggagccatgtatgctgatgacacagctggttgggacacaagaataacagaagatgacctgcacaatgaggaaaagatcatacagcaaatggaccctgaacacaggcagttagcgaacgctatattcaagctcacataccaaaacaaagtggtcaaagttcaacgaccgactccaacgggcacggtaatggatattatatctaggaaagaccaaaggggcagtggacaactgggaacttatggcctgaatacattcaccaacatggaagcccagttagtcagacaaatggaaggagaaggtgtgctgacaaaggcagacctcgagaaccctcatctgctagagaagaaaatcacacaatggttggaaaccaaaggagtggagaggttaaaaagaatggccattagcggggatgattgcgtggtgaaaccaatcgatgacaggttcgctaatgccctgcttgctttgaacgatatgggaaaggttcggaaagacatacctcaatggcagccatcaaagggatggcatgattggcaacaggttcctttctgctcccaccactttcatgaattgatcatgaaagatggaagaaagttggtggttccctgcagaccccaggacgaactaataggaagagcaagaatctctcaaggagcgggatggagccttagagaaactgcatgtctggggaaagcctacgcccaaatgtggagtctcatgtattttcacagaagagatctcagattagcatccaacgccatatgttcagcagtaccagtccactgggttcccacaagtagaacgacatggtctattcatgctcaccatcagtggatgactacagaagacatgcttactgtttggaacagggtgtggatagaggaaaatccatggatggaagacaaaactccagttacaacttgggaaaatgttccatatctaggaaagagagaagaccaatggtgtggatcacttattggtctcacttccagagcaacctgggcccagaacatacccacagcaattcaacaggtgagaagccttataggcaatgaagagttcctggactacatgccttcaatgaagagattcaggaaggaagaggagtcggagggagccatttggtaaacgtaggaagtggaaaagaggctaactgtcaggccaccttaagccacagtacggaagaagctgtgctgcctgtgagccccgtccaaggacgttaaaagaagaagtcaggccccaaagccacggtttgagcaaaccgtgctgcctgtagctccgtcgtggggacgtaaaacctgggaggctgcaaactgtggaagctgtacgcacggtgtagcagactagcggttagaggagacccctcccatgacacaacgcagcagcggggcccgagcactgagggaagctgtacctccttgcaaaggactagaggttagaggagaccccccgcaaataaaaacagcatattgacgctgggagagaccagagatcctgctgtctcctcagcatcattccaggcacagaacgccagaaaatggaatggtgctgttgaatcaac aggttct DEN-4MNQRKKVVRPPFNMLKRERNRVSTPQGLVKRFSTGLFSGKGPLRMVLAFI 21 (NC_002640.1)TFLRVLSIPPTAGILKRWGQLKKNKAIKILIGFRKEIGRMLNILNGRKRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLGEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSGMGLETRAETWMSSEGAWKHAQRVESWILRNPGFALLAGFMAYMIGQTGIQRTVFFVLMMLVAPSYGMRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFSCSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDTSNHGVTAMITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGNSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGFLVLWIGTNSRNTSMAMTCIAVGGITLFLGFTVQADMGCVASWSGKELKCGSGIFVVDNVHTWTEQYKFQPESPARLASAILNAHKDGVCGIRSTTRLENVMWKQITNELNYVLWEGGHDLTVVAGDVKGVLTKGKRALTPPVSDLKYSWKTWGKAKIFTPEARNSTFLIDGPDTSECPNERRAWNSLEVEDYGFGMFTTNIWMKFREGSSEVCDHRLMSAAIKDQKAVHADMGYWIESSKNQTWQIEKASLIEVKTCLWPKTHTLWSNGVLESQMLIPKSYAGPFSQHNYRQGYATQTVGPWHLGKLEIDFGECPGTTVTIQEDCDHRGPSLRTTTASGKLVTQWCCRSCTMPPLRFLGEDGCWYGMEIRPLSEKEENMVKSQVTAGQGTSETFSMGLLCLTLFVEECLRRRVTRKHMILVVVITLCAIILGGLTWMDLLRALIMLGDTMSGRIGGQIHLAIMAVFKMSPGYVLGVFLRKLTSRETALMVIGMAMTTVLSIPHDLMELIDGISLGLILLKIVTQFDNTQVGTLALSLTFIRSTMPLVMAWRTIMAVLFVVTLIPLCRTSCLQKQSHWVEITALILGAQALPVYLMTLMKGASRRSWPLNEGIMAVGLVSLLGSALLKNDVPLAGPMVAGGLLLAAYVMSGSSADLSLEKAANVQWDEMADITGSSPIVEVKQDEDGSFSIRDVEETNMITLLVKLALITVSGLYPLAIPVTMTLWYMWQVKTQRSGALWDVPSPAATKKAALSEGVYRIMQRGLFGKTQVGVGIHMEGVFHTMWHVTRGSVICHETGRLEPSWADVRNDMISYGGGWRLGDKWDKEEDVQVLAIEPGKNPKHVQTKPGLFKTLTGEIGAVTLDFKPGTSGSPIINRKGKVIGLYGNGVVTKSGDYVSAITQAERIGEPDYEVDEDIFRKKRLTIMDLHPGAGKTKRILPSIVREALKRRLRTLILAPTRVVAAEMEEALRGLPIRYQTPAVKSEHTGREIVDLMCHATFTTRLLSSTRVPNYNLIVMDEAHFTDPSSVAARGYISTRVEMGEAAAIFMTATPPGATDPFPQSNSPIEDIEREIPERSWNTGFDWITDYQGKTVWFVPSIKAGNDIANCLRKSGKKVIQLSRKTFDTEYPKTKLTDWDFVVTTDISEMGANFRAGRVIDPRRCLKPVILPDGPERVILAGPIPVTPASAAQRRGRIGRNPAQEDDQYVFSGDPLKNDEDHAHWTEAKMLLDNIYTPEGIIPTLFGPEREKTQAIDGEFRLRGEQRKTFVELMRRGDLPVWLSYKVASAGISYEDREWCFTGERNNQILEENMEVEIWTREGEKKKLRPRWLDARVYADPMALKDFKEFASGRKSITLDILTEIASLPTYLSSRAKLALDNIVMLHTTERGGRAYQHALNELPESLETLMLVALLGAMTAGIFLFFMQGKGIGKLSMGLITIAVASGLLWVAEIQPQWIAASIILEFFLMVLLIPEPEKQRTPQDNQLIYVILTILTIIGLIAANEMGLIEKTKTDFGFYQVKTETTILDVDLRPASAWTLYAVATTILTPMLRHTIENTSANLSLAAIANQAAVLMGLGKGWPLHRMDLGVPLLAMGCYSQVNPTTLTASLVMLLVHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGITVIDLEPISYDPKFEKQLGQVMLLVLCAGQLLLMRTTWAFCEVLTLATGPILTLWEGNPGRFWNTTIAVSTANIFRGSYLAGAGLAFSLIKNAQTPRRGTGTTGETLGEKWKRQLNSLDRKEFEEYKRSGILEVDRTEAKSALKDGSKIKHAVSRGSSKIRWIVERGMVKPKGKVVDLGCGRGGWSYYMATLKNVTEVKGYTKGGPGHEEPIPMATYGWNLVKLHSGVDVFYKPTEQVDTLLCDIGESSSNPTIEEGRTLRVLKMVEPWLSSKPEFCIKVLNPYMPTVIEELEKLQRKHGGNLVRCPLSRNSTHEMYWVSGASGNIVSSVNTTSKMLLNRFTTRHRKPTYEKDVDLGAGTRSVSTETEKPDMTIIGRRLQRLQEEHKETWHYDQENPYRTWAYHGSYEAPSTGSASSMVNGVVKLLTKPWDVIPMVTQLAMTDTTPFGQQRVFKEKVDTRTPQPKPGTRMVMTTTANWLWALLGKKKNPRLCTREEFISKVRSNAAIGAVFQEEQGWTSASEAVNDSRFWELVDKERALHQEGKCESCVYNMMGKREKKLGEFGRAKGSRAIWYMWLGARFLEFEALGFLNEDHWFGRENSWSGVEGEGLHRLGYILEEIDKKDGDLMYADDTAGWDTRITEDDLQNEELITEQMAPHHKILAKAIFKLTYQNKVVKVLRPTPRGAVMDIISRKDQRGSGQVGTYGLNTFTNMEVQLIRQMEAEGVITQDDMQNPKGLKERVEKWLKECGVDRLKRMAISGDDCVVKPLDERFGTSLLFLNDMGKVRKDIPQWEPSKGWKNWQEVPFCSHHFHKIFMKDGRSLVVPCRNQDELIGRARISQGAGWSLRETACLGKAYAQMWSLMYFHRRDLRLASMAICSAVPTEWFPTSRTTWSIHAHHQWMTTEDMLKVWNRVWIEDNPNMTDKTPVHSWEDIPYLGKREDLWCGSLIGLSSRATWAKNIHTAITQVRNLIGKEEYVDYMPVMKRYSAPSESE GVL DEN-4agttgttagtctgtgtggaccgacaaggacagttccaaatcggaagcttg 22 (NC_002640.1)cttaacacagttctaacagtttgtttgaatagagagcagatctctggaaaaatgaaccaacgaaaaaaggtggttagaccacctttcaatatgctgaaacgcgagagaaaccgcgtatcaacccctcaagggttggtgaagagattctcaaccggacttttttctgggaaaggacccttacggatggtgctagcattcatcacgtttttgcgagtcctttccatcccaccaacagcagggattctgaagagatggggacagttgaagaaaaataaggccatcaagatactgattggattcaggaaggagataggccgcatgctgaacatcttgaacgggagaaaaaggtcaacgataacattgctgtgcttgattcccaccgtaatggcgttttccctcagcacaagagatggcgaacccctcatgatagtggcaaaacatgaaagggggagacctctcttgtttaagacaacagaggggatcaacaaatgcactctcattgccatggacttgggtgaaatgtgtgaggacactgtcacgtataaatgccccctactggtcaataccgaacctgaagacattgattgctggtgcaacctcacgtctacctgggtcatgtatgggacatgcacccagagcggagaacggagacgagagaagcgctcagtagctttaacaccacattcaggaatgggattggaaacaagagctgagacatggatgtcatcggaaggggcttggaagcatgctcagagagtagagagctggatactcagaaacccaggattcgcgctcttggcaggatttatggcttatatgattgggcaaacaggaatccagcgaactgtcttctttgtcctaatgatgctggtcgccccatcctacggaatgcgatgcgtaggagtaggaaacagagactttgtggaaggagtctcaggtggagcatgggtcgacctggtgctagaacatggaggatgcgtcacaaccatggcccagggaaaaccaaccttggattttgaactgactaagacaacagccaaggaagtggctctgttaagaacctattgcattgaagcctcaatatcaaacataactacggcaacaagatgtccaacgcaaggagagccttatctgaaagaggaacaggaccaacagtacatttgccggagagatgtggtagacagagggtggggcaatggctgtggcttgtttggaaaaggaggagttgtgacatgtgcgaagttttcatgttcggggaagataacaggcaatttggtccaaattgagaaccttgaatacacagtggttgtaacagtccacaatggagacacccatgcagtaggaaatgacacatccaatcatggagttacagccatgataactcccaggtcaccatcggtggaagtcaaattgccggactatggagaactaacactcgattgtgaacccaggtctggaattgactttaatgagatgattctgatgaaaatgaaaaagaaaacatggctcgtgcataagcaatggtttttggatctgcctcttccatggacagcaggagcagacacatcagaggttcactggaattacaaagagagaatggtgacatttaaggttcctcatgccaagagacaggatgtgacagtgctgggatctcaggaaggagccatgcattctgccctcgctggagccacagaagtggactccggtgatggaaatcacatgtttgcaggacatcttaagtgcaaagtccgtatggagaaattgagaatcaagggaatgtcatacacgatgtgttcaggaaagttttcaattgacaaagagatggcagaaacacagcatgggacaacagtggtgaaagtcaagtatgaaggtgctggagctccgtgtaaagtccccatagagataagagatgtaaacaaggaaaaagtggttgggcgtatcatctcatccacccctttggctgagaataccaacagtgtaaccaacatagaattagaacccccctttggggacagctacatagtgataggtgttggaaacagcgcattaacactccattggttcaggaaagggagttccattggcaagatgtttgagtccacatacagaggtgcaaaacgaatggccattctaggtgaaacagcttgggattttggttccgttggtggactgttcacatcattgggaaaggctgtgcaccaggtttttggaagtgtgtatacaaccatgtttggaggagtctcatggatgattagaatcctaattgggttcttagtgttgtggattggcacgaactcgaggaacacttcaatggctatgacgtgcatagctgttggaggaatcactctgtttctgggcttcacagttcaagcagacatgggttgtgtggcgtcatggagtgggaaagaattgaagtgtggaagcggaatttttgtggttgacaacgtgcacacttggacagaacagtacaaatttcaaccagagtccccagcgagactagcgtctgcaatattaaatgcccacaaagatggggtctgtggaattagatcaaccacgaggctggaaaatgtcatgtggaagcaaataaccaacgagctaaactatgttctctgggaaggaggacatgacctcactgtagtggctggggatgtgaagggggtgttgaccaaaggcaagagagcactcacacccccagtgagtgatctgaaatattcatggaagacatggggaaaagcaaaaatcttcaccccagaagcaagaaatagcacatttttaatagacggaccagacacctctgaatgccccaatgaacgaagagcatggaactctcttgaggtggaagactatggatttggcatgttcacgaccaacatatggatgaaattccgagaaggaagttcagaagtgtgtgaccacaggttaatgtcagctgcaattaaagatcagaaagctgtgcatgctgacatgggttattggatagagagctcaaaaaaccagacctggcagatagagaaagcatctcttattgaagtgaaaacatgtctgtggcccaagacccacacactgtggagcaatggagtgctggaaagccagatgctcattccaaaatcatatgcgggccctttttcacagcacaattaccgccagggctatgccacgcaaaccgtgggcccatggcacttaggcaaattagagatagactttggagaatgccccggaacaacagtcacaattcaggaggattgtgaccatagaggcccatctttgaggaccaccactgcatctggaaaactagtcacgcaatggtgctgccgctcctgcacgatgcctcccttaaggttcttgggagaagatgggtgctggtatgggatggagattaggcccttgagtgaaaaagaagagaacatggtcaaatcacaggtgacggccggacagggcacatcagaaactttttctatgggtctgttgtgcctgaccttgtttgtggaagaatgcttgaggagaagagtcactaggaaacacatgatattagttgtggtgatcactctttgtgctatcatcctgggaggcctcacatggatggacttactacgagccctcatcatgttgggggacactatgtctggtagaataggaggacagatccacctagccatcatggcagtgttcaagatgtcaccaggatacgtgctgggtgtgtttttaaggaaactcacttcaagagagacagcactaatggtaataggaatggccatgacaacggtgctttcaattccacatgaccttatggaactcattgatggaatatcactgggactaattttgctaaaaatagtaacacagtttgacaacacccaagtgggaaccttagctctttccttgactttcataagatcaacaatgccattggtcatggcttggaggaccattatggctgtgttgtttgtggtcacactcattcctttgtgcaggacaagctgtcttcaaaaacagtctcattgggtagaaataacagcactcatcctaggagcccaagctctgccagtgtacctaatgactcttatgaaaggagcctcaagaagatcttggcctcttaacgagggcataatggctgtgggtttggttagtctcttaggaagcgctcttttaaagaatgatgtccctttagctggcccaatggtggcaggaggcttacttctggcggcttacgtgatgagtggtagctcagcagatctgtcactagagaaggccgccaacgtgcagtgggatgaaatggcagacataacaggctcaagcccaatcgtagaagtgaagcaggatgaagatggctctttctccatacgggacgtcgaggaaaccaatatgataacccttttggtgaaactggcactgataacagtgtcaggtctctaccccttggcaattccagtcacaatgaccttatggtacatgtggcaagtgaaaacacaaagatcaggagccctgtgggacgtcccctcacccgctgccactaaaaaagccgcactgtctgaaggagtgtacaggatcatgcaaagagggttattcgggaaaactcaggttggagtagggatacacatggaaggtgtatttcacacaatgtggcatgtaacaagaggatcagtgatctgccacgagactgggagattggagccatcttgggctgacgtcaggaatgacatgatatcatacggtgggggatggaggcttggagacaaatgggacaaagaagaagacgttcaggtcctcgccatagaaccaggaaaaaatcctaaacatgtccaaacgaaacctggccttttcaagaccctaactggagaaattggagcagtaacattagatttcaaacccggaacgtctggttctcccatcatcaacaggaaaggaaaagtcatcggactctatggaaatggagtagttaccaaatcaggtgattacgtcagtgccataacgcaagccgaaagaattggagagccagattatgaagtggatgaggacatttttcgaaagaaaagattaactataatggacttacaccccggagctggaaagacaaaaagaattcttccatcaatagtgagagaagccttaaaaaggaggctacgaactttgattttagctcccacgagagtggtggcggccgagatggaagaggccctacgtggactgccaatccgttatcagaccccagctgtgaaatcagaacacacaggaagagagattgtagacctcatgtgtcatgcaaccttcacaacaagacttttgtcatcaaccagggttccaaattacaaccttatagtgatggatgaagcacatttcaccgatccttctagtgtcgcggctagaggatacatctcgaccagggtggaaatgggagaggcagcagccatcttcatgaccgcaacccctcccggagcgacagatccctttccccagagcaacagcccaatagaagacatcgagagggaaattccggaaaggtcatggaacacagggttcgactggataacagactaccaagggaaaactgtgtggtttgttcccagcataaaagctggaaatgacattgcaaattgtttgagaaagtcgggaaagaaagttatccagttgagtaggaaaacctttgatacagagtatccaaaaacgaaactcacggactgggactttgtggtcactacagacatatctgaaatgggggccaattttagagccgggagagtgatagaccctagaagatgcctcaagccagttatcctaccagatgggccagagagagtcattttagcaggtcctattccagtgactccagcaagcgctgctcagagaagagggcgaataggaaggaacccagcacaagaagacgaccaatacgttttctccggagacccactaaaaaatgatgaagatcatgcccactggacagaagcaaagatgctgcttgacaatatctacaccccagaagggatcattccaacattgtttggtccggaaagggaaaaaacccaagccattgatggagagtttcgcctcagaggggaacaaaggaagacttttgtggaattaatgaggagaggagaccttccggtgtggctgagctataaggtagcttctgctggcatttcttacgaagatcgggaatggtgcttcacaggggaaagaaataaccaaattttagaagaaaacatggaggttgaaatttggactagagagggagaaaagaaaaagctaaggccaagatggttagatgcacgtgtatacgctgaccccatggctttgaaggatttcaaggagtttgccagtggaaggaagagtataactctcgacatcctaacagagattgccagtttgccaacttacctttcctctagggccaagctcgcccttgataacatagtcatgctccacacaacagaaagaggagggagggcctatcaacacgccctgaacgaacttccggagtcactggaaacactcatgcttgtagctttactaggtgctatgacagcaggcatcttcctgtttttcatgcaagggaaaggaatagggaaattgtcaatgggtttgataaccattgcggtggctagtggcttgctctgggtagcagaaattcaaccccagtggatagcggcctcaatcatactagagttttttctcatggtactgttgataccggaaccagaaaaacaaaggaccccacaagacaatcaattgatctacgtcatattgaccattctcaccatcattggtctaatagcagccaacgagatggggctgattgaaaaaacaaaaacggattttgggttttaccaggtaaaaacagaaaccaccatcctcgatgtggacttgagaccagcttcagcatggacgctctatgcagtagccaccacaattctgactcccatgctgagacacaccatagaaaacacgtcggccaacctatctctagcagccattgccaaccaggcagccgtcctaatggggcttggaaaaggatggccgctccacagaatggacctcggtgtgccgctgttagcaatgggatgctattctcaagtgaacccaacaaccttgacagcatccttagtcatgcttttagtccattatgcaataataggcccaggattgcaggcaaaagccacaagagaggcccagaaaaggacagctgctgggatcatgaaaaatcccacagtggacgggataacagtaatagatctagaaccaatatcctatgacccaaaatttgaaaagcaattagggcaggtcatgctactagtcttgtgtgctggacaactactcttgatgagaacaacatgggctttctgtgaagtcttgactttggccacaggaccaatcttgaccttgtgggagggcaacccgggaaggttttggaacacgaccatagccgtatccaccgccaacattttcaggggaagttacttggcgggagctggactggctttttcactcataaagaatgcacaaacccctaggaggggaactgggaccacaggagagacactgggagagaagtggaagagacagctaaactcattagacagaaaagagtttgaagagtataaaagaagtggaatactagaagtggacaggactgaagccaagtctgccctgaaagatgggtctaaaatcaagcatgcagtatcaagagggtccagtaagatcagatggattgttgagagagggatggtaaagccaaaagggaaagttgtagatcttggctgtgggagaggaggatggtcttattacatggcgacactcaagaacgtgactgaagtgaaagggtatacaaaaggaggtccaggacatgaagaaccgattcccatggctacttatggttggaatttggtcaaactccattcaggggttgacgtgttctacaaacccacagagcaagtggacaccctgctctgtgatattggggagtcatcttctaatccaacaatagaggaaggaagaacattaagagttttgaagatggtggagccatggctctcttcaaaacctgaattctgcatcaaagtccttaacccctacatgccaacagtcatagaagagctggagaaactgcagagaaaacatggtgggaaccttgtcagatgcccgctgtccaggaactccacccatgagatgtattgggtgtcaggagcgtcgggaaacattgtgagctctgtgaacacaacatcaaagatgttgttgaacaggttcacaacaaggcataggaaacccacttatgagaaggacgtagatcttggggcaggaacgagaagtgtctccactgaaacagaaaaaccagacatgacaatcattgggagaaggcttcagcgattgcaagaagagcacaaagaaacctggcattatgatcaggaaaacccatacagaacctgggcgtatcatggaagctatgaagctccttcgacaggctctgcatcctccatggtgaacggggtggtaaaactgctaacaaaaccctgggatgtgattccaatggtgactcagttagccatgacagatacaaccccttttgggcaacaaagagtgttcaaagagaaggtggataccagaacaccacaaccaaaacccggtacacgaatggttatgaccacgacagccaattggctgtgggccctccttggaaagaagaaaaatcccagactgtgcacaagggaagagttcatctcaaaagttagatcaaacgcagccataggcgcagtctttcaggaagaacagggatggacatcagccagtgaagctgtgaatgacagccggttttgggaactggttgacaaagaaagggccctacaccaggaagggaaatgtgaatcgtgtgtctataacatgatgggaaaacgtgagaaaaagttaggagagtttggcagagccaagggaagccgagcaatctggtacatgtggctgggagcgcggtttctggaatttgaagccctgggttttttgaatgaagatcactggtttggcagagaaaattcatggagtggagtggaaggggaaggtctgcacagattgggatatatcctggaggagatagacaagaaggatggagacctaatgtatgctgatgacacagcaggctgggacacaagaatcactgaggatgaccttcaaaatgaggaactgatcacggaacagatggctccccaccacaagatcctagccaaagccattttcaaactaacctatcaaaacaaagtggtgaaagtcctcagacccacaccgcggggagcggtgatggatatcatatccaggaaagaccaaagaggtagtggacaagttggaacatatggtttgaacacattcaccaacatggaagttcaactcatccgccaaatggaagctgaaggagtcatcacacaagatgacatgcagaacccaaaagggttgaaagaaagagttgagaaatggctgaaagagtgtggtgtcgacaggttaaagaggatggcaatcagtggagacgattgcgtggtgaagcccctagatgagaggtttggcacttccctcctcttcttgaacgacatgggaaaggtgaggaaagacattccgcagtgggaaccatctaagggatggaaaaactggcaagaggttcctttttgctcccaccactttcacaagatctttatgaaggatggccgctcactagttgttccatgtagaaaccaggatgaactgatagggagagccagaatctcgcagggagctggatggagcttaagagaaacagcctgcctgggcaaagcttacgcccagatgtggtcgcttatgtacttccacagaagggatctgcgtttagcctccatggccatatgctcagcagttccaacggaatggtttccaacaagcagaacaacatggtcaatccacgctcatcaccagtggatgaccactgaagatatgctcaaagtgtggaacagagtgtggatagaagacaaccctaatatgactgacaagactccagtccattcgtgggaagatataccttacctagggaaaagagaggatttgtggtgtggatccctgattggactttcttccagagccacctgggcgaagaacattcatacggccataacccaggtcaggaacctgatcggaaaagaggaatacgtggattacatgccagtaatgaaaagatacagtgctccttcagagagtgaaggagttctgtaattaccaacaacaaacaccaaaggctattgaagtcaggccacttgtgccacggtttgagcaaaccgtgctgcctgtagctccgccaataatgggaggcgtaataatccccagggaggccatgcgccacggaagctgtacgcgtggcatattggactagcggttagaggagacccctcccatcactgataaaacgcagcaaaagggggcccgaagccaggaggaagctgtactcctggtggaaggactagaggttagaggagacccccccaacacaaaaacagcatattgacgctgggaaagaccagagatcctgctgtctctgcaacatcaatccaggcacagagcgccgcaagatggattggtgttgttgatccaacaggttct

Example 19 Dengue Virus RNA Vaccine Immunogenicity in Mice

This study provides a preliminary analysis of the immunogenicity of anucleic acid mRNA vaccine using a dengue virus (DENV) serotype 2 antigenin BALB/c mice. The study utilizes 44 groups of 10 BALB/c female (5) andmale (5) mice (440 total, 6-8 weeks of age at study initiation, seeTable 10 for design summary). In this study, construct numbers used arereferenced and found in Table 14.

TABLE 14 Dengue Antigen polynucleotides ORF mRNA Protein Construct SEQSEQ SEQ Number Gene ID Description ID NO ID NO ID NO Construct 1 131502Dengue 2, 24 25 23 DEN2_D2Y98P_PrME_Hs3 D2Y98P strain, PrMEtransmembrane antigen 2 131503 Dengue 2, 27 28 26 DEN2_D2Y98P_PrME80_Hs3D2Y98P strain, PrME secreted antigen 3 131507 Dengue 2, 30 31 29DEN2_D2Y98P_PrME80_ScFv.aDEC205.FLAG_Hs3 D2Y98P strain, PrME secretedantigen with dendritic targeting ScFv against mouse DEC205 4 120554Dengue strain 2 33 34 32 DEN2_DIII_Ferritin_Hs3 domain 3 ferritinThe sequences are shown below:

TABLE 15 SEQ ID Name Sequence NOMDAMKRGLCCVLLLCGAVFVSPFHLTTRNGEPHMIVSRQEKGKSLLFKTE 23NGVNMCTLMAMDLGELCEDTITYNCPLLRQNEPEDIDCWCNSTSTWVTYGTCTATGEHRREKRSVALVPHVGMGLETRTETWMSSEGAWKHAQRIETWVLRHPGFTIMAAILAYTIGTTYFQRVLIFILLTAVAPSMTMRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKHPATLRKYCIEAKLTNTTTASRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTCKKNMEGKIVQPENLEYTIVITPHSGEEGNDTGKHGKEIKVTPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLSWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVVITWIGMNSRSTSLSVSLVLVGVVTLYLGVMVQAATGGATGCTATGAAAAGAGGCCTGTGTTGTGTGTTGCTGTTGTGCGGAGC 24TGTGTTTGTGTCACCTTTCCACCTGACTACCCGCAATGGTGAGCCCCATATGATTGTGTCGCGCCAGGAGAAGGGGAAGTCCCTCCTGTTCAAAACTGAAAACGGCGTGAACATGTGTACCCTGATGGCCATGGACCTTGGAGAACTGTGCGAGGACACCATCACCTACAATTGTCCGCTCCTGCGCCAAAACGAACCAGAAGATATCGACTGCTGGTGCAATTCCACTTCAACCTGGGTTACCTACGGAACTTGCACCGCCACGGGAGAACACAGAAGAGAAAAGCGCTCGGTGGCGCTGGTGCCTCATGTCGGAATGGGACTGGAGACTCGGACGGAGACTTGGATGTCCTCGGAGGGAGCATGGAAACATGCCCAACGGATCGAAACTTGGGTGCTGAGGCACCCTGGATTCACCATCATGGCAGCGATCCTCGCCTACACTATAGGTACTACCTACTTTCAAAGGGTGCTGATCTTCATTCTCCTCACCGCAGTGGCCCCTTCAATGACCATGAGGTGCATTGGGATCTCGAACCGGGACTTCGTCGAAGGAGTGTCCGGAGGTAGCTGGGTCGACATCGTCCTGGAACACGGAAGCTGCGTGACTACTATGGCGAAGAACAAGCCAACCTTGGACTTCGAGCTTATCAAGACCGAGGCGAAGCACCCGGCCACTCTGAGAAAGTACTGCATCGAGGCTAAGCTCACCAACACGACCACTGCCTCGCGATGCCCAACTCAGGGAGAACCGTCACTGAACGAAGAACAGGATAAACGCTTTGTGTGCAAGCATAGCATGGTGGATAGAGGCTGGGGAAACGGCTGTGGACTCTTCGGAAAGGGTGGAATTGTGACGTGCGCAATGTTCACTTGCAAGAAGAATATGGAAGGGAAGATCGTCCAGCCGGAGAACCTGGAATACACTATCGTGATCACCCCGCACTCAGGCGAGGAGAACGCAGTGGGCAACGATACCGGGAAGCACGGGAAGGAAATCAAGGTGACCCCGCAGTCGTCCATTACCGAGGCCGAACTCACCGGATACGGCACTGTGACTATGGAATGCTCGCCACGGACCGGGCTGGATTTCAATGAGATGGTGCTCTTGCAAATGGAGAACAAAGCCTGGCTGGTCCACCGCCAGTGGTTCCTCGACCTCCCCCTTCCGTGGCTGCCGGGAGCTGACACCCAAGGATCCAACTGGATCCAAAAAGAAACCCTTGTCACGTTTAAGAATCCACATGCCAAAAAGCAGGACGTGGTCGTGCTCGGAAGCCAGGAAGGAGCCATGCACACTGCGCTGACTGGAGCAACCGAAATTCAAATGTCGAGCGGCAACCTCCTCTTCACTGGACATCTGAAGTGCCGGCTGCGCATGGACAAACTGCAACTTAAGGGCATGTCATACTCGATGTGTACCGGCAAATTCAAGGTGGTGAAGGAGATCGCGGAGACTCAGCACGGGACCATCGTCATCCGGGTCCAGTATGAGGGTGATGGTTCCCCCTGCAAGATCCCTTTCGAAATCATGGATCTGGAGAAACGTCACGTGCTGGGCCGGCTGATCACTGTGAATCCGATCGTTACGGAGAAAGACAGCCCGGTGAACATCGAAGCTGAACCGCCGTTTGGGGATAGCTACATTATCATCGGCGTGGAACCAGGCCAGCTCAAGTTGTCGTGGTTCAAGAAAGGATCCAGCATCGGACAGATGTTCGAAACCACTATGCGCGGAGCCAAACGCATGGCTATCCTGGGGGACACGGCCTGGGACTTCGGGTCGCTGGGTGGTGTGTTCACCTCCATTGGAAAGGCGCTCCATCAGGTGTTTGGTGCGATCTACGGCGCCGCATTCTCCGGAGTGTCATGGACCATGAAGATCCTCATCGGAGTCGTCATCACCTGGATCGGCATGAATTCTCGGTCCACTTCCTTGAGCGTCAGCCTGGTGCTGGTCGGAGTTGTGACTCTGTACCTTGGAGTGATGGTCCAGGCCGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUG 25GAUGCUAUGAAAAGAGGCCUGUGUUGUGUGUUGCUGUUGUGCGGAGCUGUGUUUGUGUCACCUUUCCACCUGACUACCCGCAAUGGUGAGCCCCAUAUGAUUGUGUCGCGCCAGGAGAAGGGGAAGUCCCUCCUGUUCAAAACUGAAAACGGCGUGAACAUGUGUACCCUGAUGGCCAUGGACCUUGGAGAACUGUGCGAGGACACCAUCACCUACAAUUGUCCGCUCCUGCGCCAAAACGAACCAGAAGAUAUCGACUGCUGGUGCAAUUCCACUUCAACCUGGGUUACCUACGGAACUUGCACCGCCACGGGAGAACACAGAAGAGAAAAGCGCUCGGUGGCGCUGGUGCCUCAUGUCGGAAUGGGACUGGAGACUCGGACGGAGACUUGGAUGUCCUCGGAGGGAGCAUGGAAACAUGCCCAACGGAUCGAAACUUGGGUGCUGAGGCACCCUGGAUUCACCAUCAUGGCAGCGAUCCUCGCCUACACUAUAGGUACUACCUACUUUCAAAGGGUGCUGAUCUUCAUUCUCCUCACCGCAGUGGCCCCUUCAAUGACCAUGAGGUGCAUUGGGAUCUCGAACCGGGACUUCGUCGAAGGAGUGUCCGGAGGUAGCUGGGUCGACAUCGUCCUGGAACACGGAAGCUGCGUGACUACUAUGGCGAAGAACAAGCCAACCUUGGACUUCGAGCUUAUCAAGACCGAGGCGAAGCACCCGGCCACUCUGAGAAAGUACUGCAUCGAGGCUAAGCUCACCAACACGACCACUGCCUCGCGAUGCCCAACUCAGGGAGAACCGUCACUGAACGAAGAACAGGAUAAACGCUUUGUGUGCAAGCAUAGCAUGGUGGAUAGAGGCUGGGGAAACGGCUGUGGACUCUUCGGAAAGGGUGGAAUUGUGACGUGCGCAAUGUUCACUUGCAAGAAGAAUAUGGAAGGGAAGAUCGUCCAGCCGGAGAACCUGGAAUACACUAUCGUGAUCACCCCGCACUCAGGCGAGGAGAACGCAGUGGGCAACGAUACCGGGAAGCACGGGAAGGAAAUCAAGGUGACCCCGCAGUCGUCCAUUACCGAGGCCGAACUCACCGGAUACGGCACUGUGACUAUGGAAUGCUCGCCACGGACCGGGCUGGAUUUCAAUGAGAUGGUGCUCUUGCAAAUGGAGAACAAAGCCUGGCUGGUCCACCGCCAGUGGUUCCUCGACCUCCCCCUUCCGUGGCUGCCGGGAGCUGACACCCAAGGAUCCAACUGGAUCCAAAAAGAAACCCUUGUCACGUUUAAGAAUCCACAUGCCAAAAAGCAGGACGUGGUCGUGCUCGGAAGCCAGGAAGGAGCCAUGCACACUGCGCUGACUGGAGCAACCGAAAUUCAAAUGUCGAGCGGCAACCUCCUCUUCACUGGACAUCUGAAGUGCCGGCUGCGCAUGGACAAACUGCAACUUAAGGGCAUGUCAUACUCGAUGUGUACCGGCAAAUUCAAGGUGGUGAAGGAGAUCGCGGAGACUCAGCACGGGACCAUCGUCAUCCGGGUCCAGUAUGAGGGUGAUGGUUCCCCCUGCAAGAUCCCUUUCGAAAUCAUGGAUCUGGAGAAACGUCACGUGCUGGGCCGGCUGAUCACUGUGAAUCCGAUCGUUACGGAGAAAGACAGCCCGGUGAACAUCGAAGCUGAACCGCCGUUUGGGGAUAGCUACAUUAUCAUCGGCGUGGAACCAGGCCAGCUCAAGUUGUCGUGGUUCAAGAAAGGAUCCAGCAUCGGACAGAUGUUCGAAACCACUAUGCGCGGAGCCAAACGCAUGGCUAUCCUGGGGGACACGGCCUGGGACUUCGGGUCGCUGGGUGGUGUGUUCACCUCCAUUGGAAAGGCGCUCCAUCAGGUGUUUGGUGCGAUCUACGGCGCCGCAUUCUCCGGAGUGUCAUGGACCAUGAAGAUCCUCAUCGGAGUCGUCAUCACCUGGAUCGGCAUGAAUUCUCGGUCCACUUCCUUGAGCGUCAGCCUGGUGCUGGUCGGAGUUGUGACUCUGUACCUUGGAGUGAUGGUCCAGGCCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAA GUCUGAGUGGGCGGCMDAMKRGLCCVLLLCGAVFVSPFHLTTRNGEPHMIVSRQEKGKSLLFKTE 26NGVNMCTLMAMDLGELCEDTITYNCPLLRQNEPEDIDCWCNSTSTWVTYGTCTATGEHRREKRSVALVPHVGMGLETRTETWMSSEGAWKHAQRIETWVLRHPGFTIMAAILAYTIGTTYFQRVLIFILLTAVAPSMTMRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKHPATLRKYCIEAKLTNTTTASRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTCKKNMEGKIVQPENLEYTIVITPHSGEEGNDTGKHGKEIKVTPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLSWFKKGATGGATGCTATGAAAAGAGGCCTGTGTTGTGTGTTGCTGTTGTGCGGAGC 27TGTGTTTGTGTCACCTTTCCACCTGACTACCCGCAATGGTGAGCCCCATATGATTGTGTCGCGCCAGGAGAAGGGGAAGTCCCTCCTGTTCAAAACTGAAAACGGCGTGAACATGTGTACCCTGATGGCCATGGACCTTGGAGAACTGTGCGAGGACACCATCACCTACAATTGTCCGCTCCTGCGCCAAAACGAACCAGAAGATATCGACTGCTGGTGCAATTCCACTTCAACCTGGGTTACCTACGGAACTTGCACCGCCACGGGAGAACACAGAAGAGAAAAGCGCTCGGTGGCGCTGGTGCCTCATGTCGGAATGGGACTGGAGACTCGGACGGAGACTTGGATGTCCTCGGAGGGAGCATGGAAACATGCCCAACGGATCGAAACTTGGGTGCTGAGGCACCCTGGATTCACCATCATGGCAGCGATCCTCGCCTACACTATAGGTACTACCTACTTTCAAAGGGTGCTGATCTTCATTCTCCTCACCGCAGTGGCCCCTTCAATGACCATGAGGTGCATTGGGATCTCGAACCGGGACTTCGTCGAAGGAGTGTCCGGAGGTAGCTGGGTCGACATCGTCCTGGAACACGGAAGCTGCGTGACTACTATGGCGAAGAACAAGCCAACCTTGGACTTCGAGCTTATCAAGACCGAGGCGAAGCACCCGGCCACTCTGAGAAAGTACTGCATCGAGGCTAAGCTCACCAACACGACCACTGCCTCGCGATGCCCAACTCAGGGAGAACCGTCACTGAACGAAGAACAGGATAAACGCTTTGTGTGCAAGCATAGCATGGTGGATAGAGGCTGGGGAAACGGCTGTGGACTCTTCGGAAAGGGTGGAATTGTGACGTGCGCAATGTTCACTTGCAAGAAGAATATGGAAGGGAAGATCGTCCAGCCGGAGAACCTGGAATACACTATCGTGATCACCCCGCACTCAGGCGAGGAGAACGCAGTGGGCAACGATACCGGGAAGCACGGGAAGGAAATCAAGGTGACCCCGCAGTCGTCCATTACCGAGGCCGAACTCACCGGATACGGCACTGTGACTATGGAATGCTCGCCACGGACCGGGCTGGATTTCAATGAGATGGTGCTCTTGCAAATGGAGAACAAAGCCTGGCTGGTCCACCGCCAGTGGTTCCTCGACCTCCCCCTTCCGTGGCTGCCGGGAGCTGACACCCAAGGATCCAACTGGATCCAAAAAGAAACCCTTGTCACGTTTAAGAATCCACATGCCAAAAAGCAGGACGTGGTCGTGCTCGGAAGCCAGGAAGGAGCCATGCACACTGCGCTGACTGGAGCAACCGAAATTCAAATGTCGAGCGGCAACCTCCTCTTCACTGGACATCTGAAGTGCCGGCTGCGCATGGACAAACTGCAACTTAAGGGCATGTCATACTCGATGTGTACCGGCAAATTCAAGGTGGTGAAGGAGATCGCGGAGACTCAGCACGGGACCATCGTCATCCGGGTCCAGTATGAGGGTGATGGTTCCCCCTGCAAGATCCCTTTCGAAATCATGGATCTGGAGAAACGTCACGTGCTGGGCCGGCTGATCACTGTGAATCCGATCGTTACGGAGAAAGACAGCCCGGTGAACATCGAAGCTGAACCGCCGTTTGGGGATAGCTACATTATCATCGGCGTGGAACCAGGCCAGCTCAAGTTGTCGTGGTTCAAGAAAGGAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUG 28GAUGCUAUGAAAAGAGGCCUGUGUUGUGUGUUGCUGUUGUGCGGAGCUGUGUUUGUGUCACCUUUCCACCUGACUACCCGCAAUGGUGAGCCCCAUAUGAUUGUGUCGCGCCAGGAGAAGGGGAAGUCCCUCCUGUUCAAAACUGAAAACGGCGUGAACAUGUGUACCCUGAUGGCCAUGGACCUUGGAGAACUGUGCGAGGACACCAUCACCUACAAUUGUCCGCUCCUGCGCCAAAACGAACCAGAAGAUAUCGACUGCUGGUGCAAUUCCACUUCAACCUGGGUUACCUACGGAACUUGCACCGCCACGGGAGAACACAGAAGAGAAAAGCGCUCGGUGGCGCUGGUGCCUCAUGUCGGAAUGGGACUGGAGACUCGGACGGAGACUUGGAUGUCCUCGGAGGGAGCAUGGAAACAUGCCCAACGGAUCGAAACUUGGGUGCUGAGGCACCCUGGAUUCACCAUCAUGGCAGCGAUCCUCGCCUACACUAUAGGUACUACCUACUUUCAAAGGGUGCUGAUCUUCAUUCUCCUCACCGCAGUGGCCCCUUCAAUGACCAUGAGGUGCAUUGGGAUCUCGAACCGGGACUUCGUCGAAGGAGUGUCCGGAGGUAGCUGGGUCGACAUCGUCCUGGAACACGGAAGCUGCGUGACUACUAUGGCGAAGAACAAGCCAACCUUGGACUUCGAGCUUAUCAAGACCGAGGCGAAGCACCCGGCCACUCUGAGAAAGUACUGCAUCGAGGCUAAGCUCACCAACACGACCACUGCCUCGCGAUGCCCAACUCAGGGAGAACCGUCACUGAACGAAGAACAGGAUAAACGCUUUGUGUGCAAGCAUAGCAUGGUGGAUAGAGGCUGGGGAAACGGCUGUGGACUCUUCGGAAAGGGUGGAAUUGUGACGUGCGCAAUGUUCACUUGCAAGAAGAAUAUGGAAGGGAAGAUCGUCCAGCCGGAGAACCUGGAAUACACUAUCGUGAUCACCCCGCACUCAGGCGAGGAGAACGCAGUGGGCAACGAUACCGGGAAGCACGGGAAGGAAAUCAAGGUGACCCCGCAGUCGUCCAUUACCGAGGCCGAACUCACCGGAUACGGCACUGUGACUAUGGAAUGCUCGCCACGGACCGGGCUGGAUUUCAAUGAGAUGGUGCUCUUGCAAAUGGAGAACAAAGCCUGGCUGGUCCACCGCCAGUGGUUCCUCGACCUCCCCCUUCCGUGGCUGCCGGGAGCUGACACCCAAGGAUCCAACUGGAUCCAAAAAGAAACCCUUGUCACGUUUAAGAAUCCACAUGCCAAAAAGCAGGACGUGGUCGUGCUCGGAAGCCAGGAAGGAGCCAUGCACACUGCGCUGACUGGAGCAACCGAAAUUCAAAUGUCGAGCGGCAACCUCCUCUUCACUGGACAUCUGAAGUGCCGGCUGCGCAUGGACAAACUGCAACUUAAGGGCAUGUCAUACUCGAUGUGUACCGGCAAAUUCAAGGUGGUGAAGGAGAUCGCGGAGACUCAGCACGGGACCAUCGUCAUCCGGGUCCAGUAUGAGGGUGAUGGUUCCCCCUGCAAGAUCCCUUUCGAAAUCAUGGAUCUGGAGAAACGUCACGUGCUGGGCCGGCUGAUCACUGUGAAUCCGAUCGUUACGGAGAAAGACAGCCCGGUGAACAUCGAAGCUGAACCGCCGUUUGGGGAUAGCUACAUUAUCAUCGGCGUGGAACCAGGCCAGCUCAAGUUGUCGUGGUUCAAGAAAGGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAA GUCUGAGUGGGCGGCMDAMKRGLCCVLLLCGAVFVSPFHLTTRNGEPHMIVSRQEKGKSLLFKTE 29NGVNMCTLMAMDLGELCEDTITYNCPLLRQNEPEDIDCWCNSTSTWVTYGTCTATGEHRREKRSVALVPHVGMGLETRTETWMSSEGAWKHAQRIETWVLRHPGFTIMAAILAYTIGTTYFQRVLIFILLTAVAPSMTMRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKHPATLRKYCIEAKLTNTTTASRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTCKKNMEGKIVQPENLEYTIVITPHSGEEGNDTGKHGKEIKVTPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLSWFKKGGGGGSGGGGSGGGGSEVKLQQSGTEVVKPGASVKLSCKASGYIFTSYDIDWVRQTPEQGLEWIGWIFPGEGSTEYNEKFKGRATLSVDKSSSTAYMELTRLTSEDSAVYFCARGDYYRRYFDLWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSFLSTSLGNSITITCHASQNIKGWLAWYQQKSGNAPQLLIYKASSLQSGVPSRFSGSGSGTDYIFTISNLQPEDIATYYCQHYQSFPWTFGGGTKLEIKRDYKDDDDKATGGATGCTATGAAAAGAGGCCTGTGTTGTGTGTTGCTGTTGTGCGGAGC 30TGTGTTTGTGTCACCTTTCCACCTGACTACCCGCAATGGTGAGCCCCATATGATTGTGTCGCGCCAGGAGAAGGGGAAGTCCCTCCTGTTCAAAACTGAAAACGGCGTGAACATGTGTACCCTGATGGCCATGGACCTTGGAGAACTGTGCGAGGACACCATCACCTACAATTGTCCGCTCCTGCGCCAAAACGAACCAGAAGATATCGACTGCTGGTGCAATTCCACTTCAACCTGGGTTACCTACGGAACTTGCACCGCCACGGGAGAACACAGAAGAGAAAAGCGCTCGGTGGCGCTGGTGCCTCATGTCGGAATGGGACTGGAGACTCGGACGGAGACTTGGATGTCCTCGGAGGGAGCATGGAAACATGCCCAACGGATCGAAACTTGGGTGCTGAGGCACCCTGGATTCACCATCATGGCAGCGATCCTCGCCTACACTATAGGTACTACCTACTTTCAAAGGGTGCTGATCTTCATTCTCCTCACCGCAGTGGCCCCTTCAATGACCATGAGGTGCATTGGGATCTCGAACCGGGACTTCGTCGAAGGAGTGTCCGGAGGTAGCTGGGTCGACATCGTCCTGGAACACGGAAGCTGCGTGACTACTATGGCGAAGAACAAGCCAACCTTGGACTTCGAGCTTATCAAGACCGAGGCGAAGCACCCGGCCACTCTGAGAAAGTACTGCATCGAGGCTAAGCTCACCAACACGACCACTGCCTCGCGATGCCCAACTCAGGGAGAACCGTCACTGAACGAAGAACAGGATAAACGCTTTGTGTGCAAGCATAGCATGGTGGATAGAGGCTGGGGAAACGGCTGTGGACTCTTCGGAAAGGGTGGAATTGTGACGTGCGCAATGTTCACTTGCAAGAAGAATATGGAAGGGAAGATCGTCCAGCCGGAGAACCTGGAATACACTATCGTGATCACCCCGCACTCAGGCGAGGAGAACGCAGTGGGCAACGATACCGGGAAGCACGGGAAGGAAATCAAGGTGACCCCGCAGTCGTCCATTACCGAGGCCGAACTCACCGGATACGGCACTGTGACTATGGAATGCTCGCCACGGACCGGGCTGGATTTCAATGAGATGGTGCTCTTGCAAATGGAGAACAAAGCCTGGCTGGTCCACCGCCAGTGGTTCCTCGACCTCCCCCTTCCGTGGCTGCCGGGAGCTGACACCCAAGGATCCAACTGGATCCAAAAAGAAACCCTTGTCACGTTTAAGAATCCACATGCCAAAAAGCAGGACGTGGTCGTGCTCGGAAGCCAGGAAGGAGCCATGCACACTGCGCTGACTGGAGCAACCGAAATTCAAATGTCGAGCGGCAACCTCCTCTTCACTGGACATCTGAAGTGCCGGCTGCGCATGGACAAACTGCAACTTAAGGGCATGTCATACTCGATGTGTACCGGCAAATTCAAGGTGGTGAAGGAGATCGCGGAGACTCAGCACGGGACCATCGTCATCCGGGTCCAGTATGAGGGTGATGGTTCCCCCTGCAAGATCCCTTTCGAAATCATGGATCTGGAGAAACGTCACGTGCTGGGCCGGCTGATCACTGTGAATCCGATCGTTACGGAGAAAGACAGCCCGGTGAACATCGAAGCTGAACCGCCGTTTGGGGATAGCTACATTATCATCGGCGTGGAACCAGGCCAGCTCAAGTTGTCGTGGTTCAAGAAAGGAGGAGGTGGAGGATCCGGAGGCGGAGGGTCGGGCGGTGGTGGATCGGAGGTCAAACTGCAGCAATCAGGGACCGAAGTCGTGAAGCCGGGGGCTTCAGTCAAGCTGTCCTGCAAGGCCAGCGGCTATATCTTCACTAGCTACGACATCGATTGGGTGCGGCAGACTCCGGAGCAAGGACTCGAGTGGATTGGGTGGATCTTTCCGGGCGAGGGATCAACCGAGTACAACGAAAAATTTAAGGGACGGGCAACGCTGTCCGTGGACAAGAGCTCATCTACGGCGTACATGGAGCTGACGCGGCTCACGTCAGAGGATTCCGCCGTCTACTTCTGTGCCAGGGGCGACTACTACCGGCGCTACTTTGATCTGTGGGGACAAGGAACGACCGTGACTGTCTCATCAGGCGGCGGCGGATCGGGAGGAGGCGGATCGGGTGGCGGTGGTTCGGACATTCAGATGACTCAATCGCCCAGCTTCCTGTCGACCTCACTGGGGAATTCTATTACGATCACTTGTCACGCTTCGCAGAACATCAAGGGTTGGCTGGCTTGGTACCAGCAGAAAAGCGGTAACGCCCCGCAACTGCTCATCTACAAGGCATCGTCCCTGCAATCGGGAGTGCCGTCACGCTTTTCAGGATCGGGCTCCGGAACCGATTACATCTTTACCATCAGCAACCTGCAGCCGGAAGACATCGCCACTTACTACTGTCAACACTATCAGAGCTTTCCGTGGACCTTTGGAGGGGGGACCAAATTGGAGATCAAGCGCGACTACAAGGATGACGATGACAAAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUG 31GAUGCUAUGAAAAGAGGCCUGUGUUGUGUGUUGCUGUUGUGCGGAGCUGUGUUUGUGUCACCUUUCCACCUGACUACCCGCAAUGGUGAGCCCCAUAUGAUUGUGUCGCGCCAGGAGAAGGGGAAGUCCCUCCUGUUCAAAACUGAAAACGGCGUGAACAUGUGUACCCUGAUGGCCAUGGACCUUGGAGAACUGUGCGAGGACACCAUCACCUACAAUUGUCCGCUCCUGCGCCAAAACGAACCAGAAGAUAUCGACUGCUGGUGCAAUUCCACUUCAACCUGGGUUACCUACGGAACUUGCACCGCCACGGGAGAACACAGAAGAGAAAAGCGCUCGGUGGCGCUGGUGCCUCAUGUCGGAAUGGGACUGGAGACUCGGACGGAGACUUGGAUGUCCUCGGAGGGAGCAUGGAAACAUGCCCAACGGAUCGAAACUUGGGUGCUGAGGCACCCUGGAUUCACCAUCAUGGCAGCGAUCCUCGCCUACACUAUAGGUACUACCUACUUUCAAAGGGUGCUGAUCUUCAUUCUCCUCACCGCAGUGGCCCCUUCAAUGACCAUGAGGUGCAUUGGGAUCUCGAACCGGGACUUCGUCGAAGGAGUGUCCGGAGGUAGCUGGGUCGACAUCGUCCUGGAACACGGAAGCUGCGUGACUACUAUGGCGAAGAACAAGCCAACCUUGGACUUCGAGCUUAUCAAGACCGAGGCGAAGCACCCGGCCACUCUGAGAAAGUACUGCAUCGAGGCUAAGCUCACCAACACGACCACUGCCUCGCGAUGCCCAACUCAGGGAGAACCGUCACUGAACGAAGAACAGGAUAAACGCUUUGUGUGCAAGCAUAGCAUGGUGGAUAGAGGCUGGGGAAACGGCUGUGGACUCUUCGGAAAGGGUGGAAUUGUGACGUGCGCAAUGUUCACUUGCAAGAAGAAUAUGGAAGGGAAGAUCGUCCAGCCGGAGAACCUGGAAUACACUAUCGUGAUCACCCCGCACUCAGGCGAGGAGAACGCAGUGGGCAACGAUACCGGGAAGCACGGGAAGGAAAUCAAGGUGACCCCGCAGUCGUCCAUUACCGAGGCCGAACUCACCGGAUACGGCACUGUGACUAUGGAAUGCUCGCCACGGACCGGGCUGGAUUUCAAUGAGAUGGUGCUCUUGCAAAUGGAGAACAAAGCCUGGCUGGUCCACCGCCAGUGGUUCCUCGACCUCCCCCUUCCGUGGCUGCCGGGAGCUGACACCCAAGGAUCCAACUGGAUCCAAAAAGAAACCCUUGUCACGUUUAAGAAUCCACAUGCCAAAAAGCAGGACGUGGUCGUGCUCGGAAGCCAGGAAGGAGCCAUGCACACUGCGCUGACUGGAGCAACCGAAAUUCAAAUGUCGAGCGGCAACCUCCUCUUCACUGGACAUCUGAAGUGCCGGCUGCGCAUGGACAAACUGCAACUUAAGGGCAUGUCAUACUCGAUGUGUACCGGCAAAUUCAAGGUGGUGAAGGAGAUCGCGGAGACUCAGCACGGGACCAUCGUCAUCCGGGUCCAGUAUGAGGGUGAUGGUUCCCCCUGCAAGAUCCCUUUCGAAAUCAUGGAUCUGGAGAAACGUCACGUGCUGGGCCGGCUGAUCACUGUGAAUCCGAUCGUUACGGAGAAAGACAGCCCGGUGAACAUCGAAGCUGAACCGCCGUUUGGGGAUAGCUACAUUAUCAUCGGCGUGGAACCAGGCCAGCUCAAGUUGUCGUGGUUCAAGAAAGGAGGAGGUGGAGGAUCCGGAGGCGGAGGGUCGGGCGGUGGUGGAUCGGAGGUCAAACUGCAGCAAUCAGGGACCGAAGUCGUGAAGCCGGGGGCUUCAGUCAAGCUGUCCUGCAAGGCCAGCGGCUAUAUCUUCACUAGCUACGACAUCGAUUGGGUGCGGCAGACUCCGGAGCAAGGACUCGAGUGGAUUGGGUGGAUCUUUCCGGGCGAGGGAUCAACCGAGUACAACGAAAAAUUUAAGGGACGGGCAACGCUGUCCGUGGACAAGAGCUCAUCUACGGCGUACAUGGAGCUGACGCGGCUCACGUCAGAGGAUUCCGCCGUCUACUUCUGUGCCAGGGGCGACUACUACCGGCGCUACUUUGAUCUGUGGGGACAAGGAACGACCGUGACUGUCUCAUCAGGCGGCGGCGGAUCGGGAGGAGGCGGAUCGGGUGGCGGUGGUUCGGACAUUCAGAUGACUCAAUCGCCCAGCUUCCUGUCGACCUCACUGGGGAAUUCUAUUACGAUCACUUGUCACGCUUCGCAGAACAUCAAGGGUUGGCUGGCUUGGUACCAGCAGAAAAGCGGUAACGCCCCGCAACUGCUCAUCUACAAGGCAUCGUCCCUGCAAUCGGGAGUGCCGUCACGCUUUUCAGGAUCGGGCUCCGGAACCGAUUACAUCUUUACCAUCAGCAACCUGCAGCCGGAAGACAUCGCCACUUACUACUGUCAACACUAUCAGAGCUUUCCGUGGACCUUUGGAGGGGGGACCAAAUUGGAGAUCAAGCGCGACUACAAGGAUGACGAUGACAAAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUG AGUGGGCGGCMDWTWILFLVAAATRVHSKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQT 32EGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILSGGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIA KSRKSATGGATTGGACCTGGATCTTGTTTCTCGTCGCCGCAGCCACTCGCGTTCA 33TAGCAAAGGAATGTCATACTCCATGTGCACGGGAAAATTCAAGGTGGTCAAAGAGATCGCGGAGACTCAGCACGGCACCATCGTCATTCGCGTGCAAACTGAAGGAGATGGATCTCCCTGCAAGATCCCGTTCGAGATCATGGACCTGGAAAAGAGACACGTCCTCGGTAGACTGATCACCGTGAACCCGATCGTGACGGAGAAGGATTCCCCGGTGAATATTGAAGCAGAGCCTCCATTTGGGGACTCATACATTATCATTGGGGTCGAGCCGGGCCAGCTGAAGCTGAATTGGTTTAAGAAGGGCTCGTCAATCGGACAGATGTTCGAAACTACTATGAGGGGTGCAAAGCGGATGGCGATCCTCTCGGGCGGAGATATCATCAAACTCCTTAACGAACAGGTGAACAAGGAGATGCAGTCCTCAAACCTTTACATGAGCATGTCGTCCTGGTGTTACACCCATAGCCTGGACGGCGCTGGATTGTTCCTGTTTGACCATGCAGCGGAGGAATACGAACACGCCAAGAAGCTCATCATCTTCCTGAACGAGAATAACGTGCCAGTGCAACTGACCTCCATCTCGGCTCCTGAGCACAAGTTCGAAGGACTCACCCAGATCTTCCAAAAGGCCTACGAACACGAACAGCACATCAGCGAATCCATCAACAATATCGTGGACCATGCTATCAAAAGCAAAGACCATGCGACCTTCAACTTCCTGCAATGGTATGTCGCCGAACAGCACGAAGAGGAGGTGCTGTTCAAGGACATTCTCGACAAAATCGAATTGATAGGGAACGAAAATCACGGTCTGTACCTGGCCGATCAATACGTGAAGGGAATTGCC AAGTCGCGGAAGTCGTGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUG 34GAUUGGACCUGGAUCUUGUUUCUCGUCGCCGCAGCCACUCGCGUUCAUAGCAAAGGAAUGUCAUACUCCAUGUGCACGGGAAAAUUCAAGGUGGUCAAAGAGAUCGCGGAGACUCAGCACGGCACCAUCGUCAUUCGCGUGCAAACUGAAGGAGAUGGAUCUCCCUGCAAGAUCCCGUUCGAGAUCAUGGACCUGGAAAAGAGACACGUCCUCGGUAGACUGAUCACCGUGAACCCGAUCGUGACGGAGAAGGAUUCCCCGGUGAAUAUUGAAGCAGAGCCUCCAUUUGGGGACUCAUACAUUAUCAUUGGGGUCGAGCCGGGCCAGCUGAAGCUGAAUUGGUUUAAGAAGGGCUCGUCAAUCGGACAGAUGUUCGAAACUACUAUGAGGGGUGCAAAGCGGAUGGCGAUCCUCUCGGGCGGAGAUAUCAUCAAACUCCUUAACGAACAGGUGAACAAGGAGAUGCAGUCCUCAAACCUUUACAUGAGCAUGUCGUCCUGGUGUUACACCCAUAGCCUGGACGGCGCUGGAUUGUUCCUGUUUGACCAUGCAGCGGAGGAAUACGAACACGCCAAGAAGCUCAUCAUCUUCCUGAACGAGAAUAACGUGCCAGUGCAACUGACCUCCAUCUCGGCUCCUGAGCACAAGUUCGAAGGACUCACCCAGAUCUUCCAAAAGGCCUACGAACACGAACAGCACAUCAGCGAAUCCAUCAACAAUAUCGUGGACCAUGCUAUCAAAAGCAAAGACCAUGCGACCUUCAACUUCCUGCAAUGGUAUGUCGCCGAACAGCACGAAGAGGAGGUGCUGUUCAAGGACAUUCUCGACAAAAUCGAAUUGAUAGGGAACGAAAAUCACGGUCUGUACCUGGCCGAUCAAUACGUGAAGGGAAUUGCCAAGUCGCGGAAGUCGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCU

Mice were vaccinated on weeks 0 and 3 via intramuscular (IM) orintradermal (ID) routes. One group remained unvaccinated and one wasadministered 10⁵ plaque-forming units (PFU) live DENV2, D2Y98P isolatevia intravenous (IV) injection as a positive control. Serum wascollected from each mouse on weeks 1, 3, and 5; bleeds on weeks 1 and 3were in-life samples (tail vein or submandibular bleeds) and week 5 willbe a terminal (intracardiac) bleed. Individual serum samples were storedat −80° C. until analysis by neutralization or microneutralizationassay. Pooled samples from each group at the week 5 time points weretested by Western blot for reactivity with viral lysate.

TABLE 16 Detailed experimental design (treatment, readouts) Vaccine (n =10, female) mice/group) Mouse Delivered Formulation/ Group Strain week 0and 3 Chemistry Route Dose Readouts 1 Female N/A N/A N/A Serum 2 BALB/cDEN2Y98-PrME N1-methyl- ID 0.4  samples 3 6-8 (construct 1pseudouridine/ IM mg/kg collected weeks from Table 14) 5-methyl- in LNPon weeks 4 of age cytosine ID 0.08 1, 3, and 5. 5 IM mg/kg Serum in LNPanalyzed 6 ID  0.016 via 7 IM mg/kg Western in LNP blot 8 N1-methyl- ID0.4  9 pseudouridine IM mg/kg in LNP 10 ID 0.08 11 IM mg/kg in LNP 11 ID 0.016 12 IM mg/kg in LNP 13 DEN2Y98-PrME80 N1-methyl- ID 0.4  14(construct 2 pseudouridine/ IM mg/kg from Table 14) 5-methyl- in LNP 15cytosine ID 0.08 16 IM mg/kg in LNP 17 ID  0.016 18 IM mg/kg in LNP 19N1-methyl- ID 0.4  20 pseudouridine IM mg/kg in LNP 21 ID 0.08 22 IMmg/kg in LNP 23 ID  0.016 24 IM mg/kg in LNP 25 DEN2Y98- N1-methyl- ID0.4  26 PrME80-DC pseudouridine/ IM mg/kg (construct 3 5-methyl- in LNP27 from Table 14) cytosine ID 0.08 28 IM mg/kg in LNP 29 ID  0.016 30 IMmg/kg in LNP 31 N1-methyl- ID 0.4  32 pseudouridine IM mg/kg in LNP 33ID 0.08 34 IM mg/kg in LNP 35 ID  0.016 36 IM mg/kg in LNP 37 DEN2-DIII-N1-methyl- ID 0.4  38 Ferritin pseudouridine IM mg/kg (construct 4 inLNP 39 from Table 14) ID 0.08 40 IM mg/kg in LNP 41 ID  0.016 42 IMmg/kg in LNP 43 Control, — IV 10⁵ PFU D2Y98P live virusSignal was detected in groups 5, 15, 39, and 44 (live virus control) bya band that appeared between 50 and 60 kDa in the Western blot data. Thedata suggests that a mRNA vaccine to a single dengue viral antigen canproduce antibody in preliminary studies.

In order to provide a Dengue vaccine having enhanced immunogenicity, RNAvaccines for concatemeric antigens were designed and tested according tothe invention. These vaccines, which have significantly enhancedactivity, in comparison to the single protein antigens described herein,are described below.

Example 20 In Silico Prediction of T Cell Epitopes for RNA VaccineDesign

Several peptide epitopes from Dengue virus were generated and tested forantigenic activity. The peptide epitopes are designed to maximize MHCpresentation. In general the process of MHC class I presentation isquite inefficient, with only 1 peptide of 10,000 degraded moleculesactually being presented. Additionally the priming of CD8 T cell withAPCs having insufficient densities of surface peptide/MHC class Icomplexes results in weak responders exhibiting impaired cytokinesecretion and a decrease memory pool. Thus, the process of designinghighly effective peptide epitopes is important to the immunogenicity ofthe ultimate vaccine.

In silico prediction of desirable peptide epitopes was performed usingImmune Epitope Database. Using this database several immunogenic DengueT cell epitopes showing strong homology across all 4 Dengue serotypeswere predicted. Examples of these epitopes are shown in FIGS. 16A-16Cand 17A-17C.

Example 21 Prediction of DENV T Cell Epitopes for RNA Vaccine Design

The design of optimized vaccination systems to prevent or treatconditions that have failed to respond to more traditional treatments orearly vaccination strategies relies on the identification of theantigens or epitopes that play a role in these conditions and which theimmune system can effectively target. T cell epitopes (e.g., MHC peptidebinding) for the various alleles shown in Table 17 were determined usingRapid Epitope Discovery System (ProImmune REVEAL & ProVE®). This systemis used to identify those candidate epitopes that actually causerelevant immune responses from the numerous other potential candidatesidentified using algorithms to predict MHC-peptide binding. The REVEALbinding assay determines the ability of each candidate peptide to bindto one or more MHC I class alleles and stabilize the MHC-peptidecomplex. The assay identifies the most likely immunogenic peptides in aprotein sequence by comparing the binding to that of a high affinity Tcell epitope and detecting the presence or absence of the nativeconformation of the MHC-peptide complex. The epitope peptides arefurther tested using the assays described herein to confirm theirimmunogenic activity.

TABLE 17 Alleles Tested Allele A*01:01 A*02:01 A*03:01 A*11:01 A*24:02B*07:02 B*27:05 H-2Kb

TABLE 18 ProImmune REVEAL ® binding assay data for A*01:01 Peptide I.D.SEQ ID NO REVEAL ® score at 0 h TTDISEMGA 217 68.4

TABLE 19 ProImmune REVEAL ® binding assay data for A*02:01 Peptide I.D.SEQ ID NO REVEAL ® score at 0 h TMWHVTRGA 218 112.0 MWHVTRGAV 219 62.7GLYGNGVVT 220 87.7 TLILAPTRV 221 104.2 LILAPTRVV 222 106.4 ILAPTRVVA 22395.7 VVAAEMEEA 224 92.2 IVDLMCHAT 225 62.7 LMCHATFTM 226 72.9 MGEAAAIFM227 50.6 GEAAAIFMT 228 74.3 KTVWFVPSI 229 115.9 LMRRGDLPV 230 82.3TLLCDIGES 231 63.9 LLCDIGESS 232 93.9 AMTDTTPFG 233 91.9 GQQRVFKEK 23447.1 KLTYQNKVV 235 92.3 AISGDDCVV 236 91.1 LMYFHRRDL 237 97.8

TABLE 20 ProImmune REVEAL ® binding assay data for A*03:01 Peptide I.D.SEQ ID NO REVEAL ® score at 0 h RTLILAPTR 238 91.4 TLILAPTRV 239 55.2MCHATFTMR 240 86.8 TVWFVPSIK 241 53.6 GQQRVFKEK 242 59.6 CVYNMMGKR 24381.6

TABLE 21 ProImmune REVEAL ® binding assay data for A*11:01 Peptide I.D.SEQ ID NO REVEAL ® score at 0 h HTMWHVTRG 244 56.3 RTLILAPTR 245 89.9TLILAPTRV 246 59.0 MCHATFTMR 247 91.0 ATFTMRLLS 248 58.5 GEAAAIFMT 24950.3 KTVWFVPSI 250 50.8 TVWFVPSIK 251 92.2 GQQRVFKEK 252 85.5 CVYNMMGKR253 113.2 VYNMMGKRE 254 62.5 YNMMGKREK 255 80.9 NMMGKREKK 256 77.9GTYGLNTFT 257 63.6 ISGDDCVVK 258 88.7

TABLE 22 ProImmune REVEAL ® binding assay data for A*24:02 Peptide I.D.SEQ ID NO REVEAL ® score at 0 h LMCHATFTM 259 99.5 CHATFTMRL 260 75.9GEAAAIFMT 261 58.9 KTVWFVPSI 262 89.1 HWTEAKMLL 263 103.2 WTEAKMLLD 26494.7 LGCGRGGWS 265 74.8 MAMTDTTPF 266 51.3 MYADDTAGW 267 76.8 VGTYGLNTF268 96.0 YFHRRDLRL 269 87.5

TABLE 23 ProImmune REVEAL ® binding assay data for B*07:02 Peptide I.D.SEQ ID NO REVEAL ® score at 0 h FKPGTSGSP 270 50.4 KPGTSGSPI 271 112.1IPERSWNSG 272 45.2 PERVILAGP 273 56.1 LMRRGDLPV 274 178.9 PLSRNSTHE 27565.0 LSRNSTHEM 276 124.5 SRNSTHEMY 277 52.0 MAMTDTTPF 278 117.4TPFGQQRVF 279 112.7 LMYFHRRDL 280 119.6

TABLE 24 ProImmune REVEAL ® binding assay data for B*27:05 Peptide I.D.SEQ ID NO REVEAL ® score at 0 h LRTLILAPT 281 58.7 LMCHATFTM 282 98.2ARGYISTRV 283 125.3 RRGDLPVWL 284 144.8 GQQRVFKEK 285 95.4 SRAIWYMWL 28653.9 FKLTYQNKV 287 53.7

TABLE 25 ProImmune REVEAL ® binding assay data for H-2Kb Peptide I.D.SEQ ID NO REVEAL ® score at 0 h FKPGTSGSP 288 45.7 LAPTRVVAA 289 102.5LMCHATFTM 290 59.0 CHATFTMRL 291 60.3 HATFTMRLL 292 69.5 ATFTMRLLS 29355.6 KTVWFVPSI 294 54.4 LSRNSTHEM 295 51.1 QQRVFKEKV 296 63.4 YGLNTFTNM297 75.4 LMYFHRRDL 298 54.9

Example 22 Activity Testing for Predicted Peptide Epitopes

Exemplary peptide epitopes selected using the methods described abovewere further characterized. These peptide epitopes were confirmed tohave activity using in vitro HLA binding assays (human lymphocytebinding assays). Peptides (9 aa peptides from the dengue antigen) werescreened for their ability to bind to HLA. The analysis of the homology,affinity, frequency and design of these peptides is shown in FIGS.16A-16C and 17A-17C.

Example 23 In Vivo Analysis of Mimectopes of Predicted Human EpitopesRNA Vaccines

Methods

IFNγ ELISpot. Mouse IFNγ ELISpot assays were performed using IFNγ coatedMillipore IP Opaque plates according to the manufacturer's mouse IFNγELISPOT guidelines. Briefly, the plates were blocked using complete RPMI(R10) and incubated for 30 minutes prior to plating cells. Peptides(284-292, 408-419 or 540-548) were diluted to 5 different concentrationsfor stimulation at 5, −6, −7, −8, or −9 from an original stockconcentration of 10 mM (−2). Mouse splenocytes (200,000-250,000 cells)were plated in appropriate wells with peptide, PMA+ Ionomycin or R10media alone. Cells were stimulated in a total volume of 125 μL per well.Plates were then incubated at 37° C., 5% CO₂ for 18-24 hrs. Plates weredeveloped following the manufacturer's instructions. Plates were countedand quality controlled using the automated ELISPOT reader CTLImmunoSpot/FluoroSpot.

Intracellular Cytokine Staining (ICS). Intracellular Cytokine Staining(ICS). For intracellular cytokine staining, individual splenocytes, wereresuspended at a concentration of 1.5×106 cells per mL. Peptides(284-292, 408-419 or 540-548) were made into 5 dilutions from a stockconcentration of 10 mM⁽⁻²⁾. The final concentrations of each peptidewere −5, −6, −7, −8, or −9 in their respective wells. Cells werestimulated in a final volume of 200 uL within a 96 well culture plate.After the addition of Golgi plug (0.2 uL per well), cells were incubatedat 37° C., 5% CO₂ for 5 hours. Following stimulation, cells were surfacestained, fixed, washed and put at 4° C. overnight. Intracellularstaining was performed the following day, resulting in full panel ofLive/Dead (Invitrogen), αCD3, αCD4, αCD8, αCD45, αCCR7, αCD44, αCD25,αIL-2, αIFNγ, and αTNFα (BD Biosciences). Cells were acquired in a 96-Ubottom plate using BD LSR Fortessa HTS (BD Biosciences).

Results

The exemplary peptide epitopes selected using the methods describedherein were used to produce tests mouse mimectopes of the predictedhuman epitopes. These mimectopes were analyzed for in vivo activityusing restimulation assays during the acute phase of Dengue infection(Day 7). The methods were performed on dengue-infectedIFNαβ/γ-receptor-deficient mice (AG129). Seven days post infectionsplenocytes were harvested and subjected to an ELISPOT assay to quantifysecretion of cytokines by T cells (CD8) as described above. Briefly, theisolated splenocytes were stimulated with the test peptides and testedfor T cell activation. If the peptide is an appropriate antigen, somecells would be present antigen during infection and would be capable ofstimulating T cells. The methods for analyzing the T cell activationwere performed as follows:

-   -   T cells (at a known concentration) were incubated with a        specific antigen in a cell culture well    -   the activated T cells were transferred to ELISPOT plates        (precoated with anti-cytokine antibody)    -   the cells were incubated such that cytokines could be secreted    -   the cells were washed off the plate and enzyme coupled secondary        Ig was added    -   the plates were washed and substrate was added    -   positive spots were scored under microscope.

The data is shown in FIGS. 18-19. FIGS. 18 and 19 are graphs depictingthe results of an ELISPOT assay of dengue-specific peptides measuringIFN-γ (spots per million splenocytes).

A schematic of an assay on a BLT Mouse Model (Bone Marrow/Liver/Thymus)is shown in FIG. 20. The results of a histogram analysis of human CD8 Tcells stimulated with peptide epitope is also shown in FIG. 20.

The following two sequences were used as controls:

(V5)8-cathb: (SEQ ID NO: 35)Kozak Start GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST Stop (v5)8-cathb + MHCi:(SEQ ID NO: 36) Kozak Start GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST-GFLG-GKPIPNPLLGLDST StopSome results are shown in Table 26:

TABLE 26 Results A*02:01 Peptide ID REVEAL ® Score 5.KQWFLDLPL (SEQ ID NO: 213) 86.0 6. RQWFLDLPL (SEQ ID NO: 214) 77.7 7.RQWFFDLPL (SEQ ID NO: 215) 80.5 8. TALTGATEI (SEQ ID NO: 216) 0.9Positive Control 100. +/−

Example 24 AG129 Mouse Challenge of Mimectopes of Predicted HumanEpitopes from DENV2

A study is performed on AG129 mouse using a cocktail of 2 peptideepitopes. The immunogenicity of the peptide epitopes is determined inAG129 mice against challenge with a lethal dose of mouse-adapted DENV 2strain D2Y98P. AG129 mice, which lack IFN α/β and γ receptor signaling,injected intradermally in the footpad with 10⁴ PFU of DENV do notsurvive past day 5 post-injection. AG129 mice are vaccinated viaintramuscular (IM) injection with either 2 μg or 10 μg of a cocktail of2 peptide epitopes. The vaccines are given to AG129 mice with a primeand a boost (day 0 and day 28). The positive control group is vaccinatedwith heat-inactivated DENV 2. Phosphate-buffered saline (PBS) is used asa negative control. On day 56, mice are challenged with mouse-adaptedDENV 2 and monitored for 10 days for weight loss, morbidity, andmortality. Mice that display severe illness, defined as >30% weightloss, a health score of 6 or above, extreme lethargy, and/or paralysisare euthanized.

Example 25 “Humanized” DENV Peptides Mouse Immunogenicity Study

A study analyzing immunogenicity of the peptide epitopes on humanizedmice is performed. A single-dose cocktail (30 μg) containing 3 differentpeptide epitopes are delivered by IM route of immunization with primeand boost (day 0, day 28). A T cell (ELISPOT and ICS) characterizationmay be performed on Day 7, Day 28, and Day 56.

Example 26 Testing of Non-Human Primate (NHP) Mimectopes of PredictedDENV Human Epitopes

Non-human primate (NHP) mimectopes to the human epitopes may also bedeveloped and tested for activity in NHP assays. The NHP mimectopes aredesigned based on the human antigen sequence. These mimectopes may beanalyzed for in vivo activity in an NHP model using, for instance,restimulation assays. Once the NHPs have been infected, immune cells maybe isolated and tested for sensitivity of activation by the particularmimectopes.

Example 27 Targeting of DENV Concatemeric Constructs Using CytoplasmicDomain of MHC I

MHC-1_V5 concatemer constructs were developed and transfected in HeLacells. Triple immunofluorescence using Mitotracker Red (mitochondria),anti-V5, and anti-MHC-1 antibodies plus Dapi was performed. The data isshown in FIGS. 21-23. FIG. 21 shows MHC-1_V5 concatemer transfection inHeLa cells. The arrows indicate V5-MHC1 colocalization (bottom right).FIG. 22 shows MHC-1_V5 concatemer transfection. The arrows indicateregions where V5 preferentially colocalizes with MHC1 and not withMitotracker. FIG. 23 shows V5 concatemer transfection in HeLa cells. V5has homogeneous cytoplasmic distribution preferentially colocalizes withMHC1 and not with Mitotracker. These data demonstrate that the V5concatemer with the cytoplasmic domain from MHC class I colocalizes withMHC class I expression (FIG. 21), while the V5 concatemer without thissequence is only found in the cytoplasm (FIG. 23) following transfectionin HeLa cells.

Example 28 In Vivo Analysis of DENV Concatemeric mRNA Epitope Construct

The Dengue concatemers used in this study consist of 8 repeats of thepeptide TALGATEI (SEQ ID NO: 299), a mouse CD8 T cell epitope found inthe DENV2 envelope. The peptide repeats were linked via cathepsin Bcleavage sites and modified with the various sequences as follows:

-   (1) TALGATEI (SEQ ID NO: 299) peptide concatemer with no    modification-   (2) TALGATEI (SEQ ID NO: 299) peptide concatemer with IgKappa signal    peptide-   (3) TALGATEI (SEQ ID NO: 299) peptide concatemer with PEST sequence-   (4) TALGATEI (SEQ ID NO: 299) peptide concatemer with IgKappa signal    peptide and PEST sequence-   (5) TALGATEI (SEQ ID NO: 299) peptide concatemer with MHC class I    cytoplasmic domain-   (6) TALGATEI (SEQ ID NO: 299) peptide concatemer with IgKappa signal    peptide and MHC class I cytoplasmic domain-   (7) Heat-inactivated DENV2 (D2Y98P)-   (8) No immunization

The immunogenicity of the peptide concatemeric candidate vaccines weredetermined in AG129 mice against challenge with a lethal dose of DENVstrain D2Y98P. AG129 mice, which lack IFN α/β and γ receptor signaling,injected intradermally in the footpad with 10⁴ PFU of DENV do notsurvive past day 5 post-injection. (In this study, the mice died due toa problem with the heat-attenuation). The tested vaccines includedconstructs (1)-(8) disclosed above. AG129 mice were vaccinated viaintramuscular (IM) injection with either 2 μg or 10 μg of the candidatevaccine. The vaccines were given to AG129 mice as a prime and a boost(second dose provided 28 days after the first dose). The positivecontrol group was vaccinated with heat-inactivated DENV 2.Phosphate-buffered saline (PBS) was used as a negative control.

On day 56, mice were challenged with mouse-adapted DENV 2 and monitoredfor 10 days for weight loss, morbidity, and mortality. Mice thatdisplayed severe illness, defined as >30% weight loss, a health score of6 or above, extreme lethargy, and/or paralysis were euthanized. Notably,mice “vaccinated” with heat-inactivated DENV (positive control group)became morbid and died (they were not included in the challenge portionof the study).

In addition, individual serum samples were collected prior to challengeon day 54 and PBMCs were isolated and frozen for subsequent testing.

The AG129 mice PBMCs were thawed and stimulated with TALGATEI (SEQ IDNO: 299) peptide for 5 hours in a standard intracellular cytokine assay.For intracellular cytokine staining, PBMCs were thawed and suspended inmedia. The TALGATEI (SEQ ID NO: 299) peptide was administered tostimulate the cells. After the addition of Golgi plug, cells wereincubated at 37° C., 5% CO2 for 5 hours. Following stimulation, cellswere surface stained, fixed, washed and put at 4° C. overnight.Intracellular staining was performed the following day and assayed viaELISPOT assay to quantify secretion of cytokines by T cells (CD8) asdescribed above to determine T cell activation. If the peptide is anappropriate antigen, some cells would be present antigen duringinfection and would be capable of stimulating T cells. The results areshown in FIGS. 24A and 24B, which demonstrate that each of the peptides(1)-(6) stimulate T cell activation.

Example 29 Exemplary CHIKV Polypeptides

The amino acids presented in the Table 27 are exemplary CHIKV antigenicpolypeptides. To the extent that any exemplary antigenic peptidedescribed herein includes a flag tag or V5, or a polynucleotide encodesa flag tag or V5, the skilled artisan understands that such flag tag orV5 is excluded from the antigenic polynucleotide in a vaccineformulation. Thus, any of the polynucleotides encoding proteinsdescribed herein are encompassed within the compositions of theinvention without the flag tag or V5 sequence.

TABLE 27 Antigen identifier Amino acid sequence SE_chikv-MYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELLSVTLEPTLSLDYITCEYKTVIPSPYVKBrazillian-CCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDTENTQLSEAHVEKSESCKTEFASAYRE1_KP164567-AHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVY 71_72NMDYPPFGAGRPGQFGDIQSRTPESEDVYANTQLVLQRPSAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFGCQIATNPVRAMNCAVGNMPISIDIPDAAFTRVVDAPSLTDMSCEVSACTHSSDFGGVAIIKYAASKKGKCAVHSMTNAVTIREAEIEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAECHPPKDHIVNYPASHTTLGVQDISATAMSWVQKITGGVGLVVAVAALILIVVLCVSFSRH (SEQ ID NO. 37) SE_chikv-MYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELLSVTLEPTLSLDYITCEYKTVIPSPYVKBrazillian-CCGTAECKDKNLPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASAYRE1_KP164568-AHTASASAKLRVLYQGNNITVTAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVY 69_70NMDYPPFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFGCQIATNPVRAVNCAVGNMPISIDIPDAAFIRVVDAPSLTDMSCEVPACTHSSDFGGVAIIKYAASKKGKCAVHSMTNAVTIREAEIEVEGNSQLQISFSTALASAEFRVQVCSTQVHCVAECHPPKDHIVNYPASHTTLGVQDISATALSWVQKITGGVGLVVAVAALILIVVLCVSFSRH (SEQ ID NO. 38) SE_chikv-MSIKDHFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSBrazillian-HDWTKLRYMDNHMPADAERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDGRKISH E2-SCTHPFHHDPPVIGREKFHSRPQHGRELPCSTYAQSTAATAEEIEVHMPPDTPDRTLMSQQSE1_KP164567-GNVKITVNSQTVRYKCNCGDSSEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAE 71_72FGDRKGKVHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVTHKKEIRLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTAVVLSVASFILLSMVGVAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELLSVTLEPTLSLDYITCEYKTVIPSPYVKCCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDTENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGRPGQFGDIQSRTPESEDVYANTQLVLQRPSAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFGCQIATNPVRAMNCAVGNMPISIDIPDAAFTRVVDAPSLTDMSCEVSACTHSSDFGGVAIIKYAASKKGKCAVHSMTNAVTIREAEIEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAECHPPKDHIVNYPASHTTLGVQDISATAMSWVQKITGGVGLVVAVAALILIVVLCVSFSRHMSIKDHFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDNHMPADAERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDGRKISHSCTHPFHHDPPVIGREKFHSRPQHGRELPCSTYAQSTAATAEEIEVHMPPDTPDRTLMSQQSGNVKITVNSQTVRYKCNCGDSSEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAEFGDRKGKVHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVTHKKEIRLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTAVVLSVASFILLSMVGVAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELLSVTLEPTLSLDYITCEYKTVIPSPYVKCCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDTENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGRPGQFGDIQSRTPESEDVYANTQLVLQRPSAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFGCQIATNPVRAMNCAVGNMPISIDIPDAAFTRVVDAPSLTDMSCEVSACTHSSDFGGVAIIKYAASKKGKCAVHSMTNAVTIREAEIEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAECHPPKDHIVNYPASHTTLGVQDISATAMSWVQKITGGVGLVVAVAALILIVVLCVSFSRH (SEQ ID NO. 39)SQ-031495 MSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSSE_chikv- HDWTKLRYMDNHTPADAERAGLFVRTSAPCTITGTMGHFILTRCPKGETLTVGFTDSRKISHBrazillian-SCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDRTLMSQQS E2-GNVKITVNGQTVRYKCNCGGSNEGLITTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAEE1_KP164568-LGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEE 69_70WVTHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVASFVLLSMVGVAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKAYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELLSVTLEPTLSLDYITCEYKTVIPSPYVKCCGTAECKDKNLPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRVLYQGNNITVTAYANGDHAVTVKDAKFIVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFGCQIATNPVRAVNCAVGNMPISIDIPDAAFIRVVDAPSLTDMSCEVPACTHSSDFGGVAIIKYAASKKGKCAVHSMTNAVTIREAEIEVEGNSQLQISFSTALASAEFRVQVCSTQVHCVAECHPPKDHIVNYPASHTTLGVQDISATALSWVQKITGGVGLVVAVAALILIVVLCVSFSRH (SEQID NO. 40) SE_chikv-MSIKDHFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSBrazillian-HDWTKLRYMDNHMPADAERAGLFVRTSAPCTITGTMGHFILARCPKGETLTVGFTDGRKISHE2_KP164567-SCTHPFHHDPPVIGREKFHSRPQHGRELPCSTYAQSTAATAEEIEVHMPPDTPDRTLMSQQS 71_72GNVKITVNSQTVRYKCNCGDSSEGLTTTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAEFGDRKGKVHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVTHKKEIRLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTAVVLSVASFILLSMVGVAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA (SEQ ID NO. 41)SE_chikv- MSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPVALERIRNEATDGTLKIQVSLQIGIKTDDSBrazillian-HDWTKLRYMDNHTPADAERAGLFVRTSAPCTITGTMGHFILTRCPKGETLTVGFTDSRKISHE2_KP164568-SCTHPFHHDPPVIGREKFHSRPQHGKELPCSTYVQSTAATTEEIEVHMPPDTPDRTLMSQQS 69_70GNVKITVNGQTVRYKCNCGGSNEGLITTDKVINNCKVDQCHAAVTNHKKWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVIMLLYPDHPTLLSYRNMGEEPNYQEEWVTHKKEVVLTVPTEGLEVTWGNNEPYKYWPQLSTNGTAHGHPHEIILYYYELYPTMTVVVVSVASFVLLSMVGVAVGMCMCARRRCITPYELTPGATVPFLLSLICCIRTAKA (SEQ ID NO. 42)SE_CHIKV_C_E3_E2_6K_E1_noMEFIPTQTFYNRRYQPRPWAPRPTIQVIRPRPRPQRQAGQLAQLISAVNKLTMRAVPQQKPRFlag or V5 orRNRKNKKQRQKKQAPQNDPKQKKQPPQKKPAQKKKKPGRRERMCMKIENDCIFEVKHEGKVMHA (StrainGYACLVGDKVMKPAHVKGTIDNADLAKLAFKRSSKYDLECAQIPVHMKSDASKFTHEKPEGY 37997YNWHHGAVQYSGGRFTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEGARTALSVVTWNKD Senegal)IVTKITPEGAEEWSLALPVLCLLANTTFPCSQPPCTPCCYEKEPESTLRMLEDNVMRPGYYQLLKASLTCSPHRQRRSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPIALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDSHTPADAERAGLLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHTCTHPFHHEPPVIGRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDTPDRTLMTQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKIDQCHAAVTNHKNWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVTMLLYPDHPTLLSYRNMGQEPNYHEEWVTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQMSTNGTAHGHPHEIILYYYELYPTMTVVIVSVASFVLLSMVGTAVGMCVCARRRCITPYELTPGATVPFLLSLLCCVRTTKAATYYEAAAYLWNEQQPLFWLQALIPLAALIVLCNCLKLLPCCCKTLAFLAVMSIGAHTVSAYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELQSVTLEPTLSLDYITCEYKTVIPSPYVKCCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFVVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFGCQIATNPVRAVNCAVGNIPISIDIPDAAFTRVVDAPSVTDMSCEVPACTHSSDFGGVAIIKYTASKKGKCAVHSMTNAVTIREADVEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAACHPPKDHIVNYPASHTTLGVQDISTTAMSWVQKITGGVGLIVAVAALILIVVLCVSFSRH (SEQ ID NO. 43) SE_CHIKV_C_E3_E2_6K_E1_noMEFIPTQTFYNRRYQPRPWAPRPTIQVIRPRPRPQRQAGQLAQLISAVNKLTMRAVPQQKPRFlag or V5 orRNRKNKKQRQKKQAPQNDPKQKKQPPQKKPAQKKKKPGRRERMCMKIENDCIFEVKHEGKVM HA_DXGYACLVGDKVMKPAHVKGTIDNADLAKLAFKRSSKYDLECAQIPVHMKSDASKFTHEKPEGYYNWHHGAVQYSGGRFTIPTGAGKPGDSGRPIFDNKGRVVAIVLGGANEGARTALSVVTWNKDIVTKITPEGAEEWSLALPVLCLLANTTFPCSQPPCTPCCYEKEPESTLRMLEDNVMRPGYYQLLKASLTCSPHRQRRSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPIALERIRNEATDGTLKIQVSLQIGIKTDDSHDWTKLRYMDSHTPADAERAGLLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISHTCTHPFHHEPPVIGRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDTPDRTLMTQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKIDQCHAAVTNHKNWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVTMLLYPDHPTLLSYRNMGQEPNYHEEWVTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQMSTNGTAHGHPHEIILYYYELYPTMTVVIVSVASFVLLSMVGTAVGMCVCARRRCITPYELTPGATVPFLLSLLCCVRTTKAATYYEAAAYLWNEQQPLFWLQALIPLAALIVLCNCLKLLPCCCKTLAFLAVMSIGAHTVSAYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELQSVTLEPTLSLDYITCEYKTVIPSPYVKCCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFVVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFGCQIATNPVRAVNCAVGNIPISIDIPDAAFTRVVDAPSVTDMSCEVPACTHSSDFGGVAIIKYTASKKGKCAVHSMTNAVTIREADVEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAACHPPKDHIVNYPASHTTLGVQDISTTAMSWVQKITGGVGLIVAVAALILIVVLCVSFSRH (SEQ ID NO. 44) SE_CHIKV_E1_noMYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELQSVTLEPTLSLDYITCEYKTVIPSPYVKFlag or V5CCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFVVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFGCQIATNPVRAVNCAVGNIPISIDIPDAAFTRVVDAPSVTDMSCEVPACTHSSDFGGVAIIKYTASKKGKCAVHSMTNAVTIREADVEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAACHPPKDHIVNYPASHTTLGVQDISTTAMSWVQKITGGVGLIVAVAALILIVVLCVSFSRH (SEQ ID NO. 45) CHIKV_E2_6K_E1_noMSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPIALERIRNEATDGTLKIQVSLQIGIKTDDS Flag orHDWTKLRYMDSHTPADAERAGLLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISH V5TCTHPFHHEPPVIGRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDTPDRTLMTQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKIDQCHAAVTNHKNWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVTMLLYPDHPTLLSYRNMGQEPNYHEEWVTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQMSTNGTAHGHPHEIILYYYELYPTMTVVIVSVASFVLLSMVGTAVGMCVCARRRCITPYELTPGATVPFLLSLLCCVRTTKAATYYEAAAYLWNEQQPLFWLQALIPLAALIVLCNCLKLLPCCCKTLAFLAVMSIGAHTVSAYEHVTVIPNTVGVPYKTLVNRPGYSPMVLEMELQSVTLEPTLSLDYITCEYKTVIPSPYVKCCGTAECKDKSLPDYSCKVFTGVYPFMWGGAYCFCDAENTQLSEAHVEKSESCKTEFASAYRAHTASASAKLRVLYQGNNITVAAYANGDHAVTVKDAKFVVGPMSSAWTPFDNKIVVYKGDVYNMDYPPFGAGRPGQFGDIQSRTPESKDVYANTQLVLQRPAAGTVHVPYSQAPSGFKYWLKERGASLQHTAPFGCQIATNPVRAVNCAVGNIPISIDIPDAAFTRVVDAPSVTDMSCEVPACTHSSDFGGVAIIKYTASKKGKCAVHSMTNAVTIREADVEVEGNSQLQISFSTALASAEFRVQVCSTQVHCAAACHPPKDHIVNYPASHTTLGVQDISTTAMSWVQKITGGVGLIVAVAALILIVVLCVSFSRH (SEQ ID NO. 46)SE_CHIKV_E2_noMSTKDNFNVYKATRPYLAHCPDCGEGHSCHSPIALERIRNEATDGTLKIQVSLQIGIKTDDS Flag orHDWTKLRYMDSHTPADAERAGLLVRTSAPCTITGTMGHFILARCPKGETLTVGFTDSRKISH V5TCTHPFHHEPPVIGRERFHSRPQHGKELPCSTYVQSTAATAEEIEVHMPPDTPDRTLMTQQSGNVKITVNGQTVRYKCNCGGSNEGLTTTDKVINNCKIDQCHAAVTNHKNWQYNSPLVPRNAELGDRKGKIHIPFPLANVTCRVPKARNPTVTYGKNQVTMLLYPDHPTLLSYRNMGQEPNYHEEWVTHKKEVTLTVPTEGLEVTWGNNEPYKYWPQMSTNGTAHGHPHEIILYYYELYPTMTVVIVSVASFVLLSMVGTAVGMCVCARRRCITPYELTPGATVPFLLSLLCCVRTTKA (SEQ ID NO. 47)

Example 30 ZIKV Vaccines

The design of preferred Zika vaccine mRNA constructs of the inventionencode prME proteins from the Zika virus intended to produce significantimmunogenicity. The open reading frame comprises a signal peptide (tooptimize expression into the endoplasmic reticulum) followed by the ZikaprME polyprotein sequence. The particular prME sequence used is from aMicronesian strain (2007) that most closely represents a consensus ofcontemporary strain prMEs. This construct has 99% prME sequence identityto the current Brazilian isolates.

Within the Zika family, there is a high level of homology within theprME sequence (>90%) across all strains so far isolated (See Table 28below). The high degree of homology is also preserved when comparing theoriginal isolates from 1947 to the more contemporary strains circulatingin Brazil in 2015, suggesting that there is “drift” occurring from theoriginal isolates. Furthermore, attenuated virus preparations haveprovided cross-immunization to all other strains tested, including LatinAmerican/Asian, and African.

Overall, this data suggests that cross-protection of all Zika strains ispossible with a vaccine based on prME.

TABLE 28 Zika virus prME homology Zika virus Pairwise AA % identityCountry of Year of to Brazilian isolates Strain Isolation Isolation prMEGenome South Suriname 2015 100.0% 99.0% American Asian Cambodia 201099.4% 99.1% French Polynesia 2013 99.7% 99.4% Micronesia 2007 98.8%97.1% African Senegal 2002 92.5% 89.9% Ugnada 1947 91.0% 87.3%

In fact, the prM/M and E proteins of ZIKV have a very high level (99%)of sequence conservation between the currently circulating Asiatic andBrazilian viral strains. The sequence alignment of the prM/M and Eproteins is shown in FIG. 27.

The M and E proteins are on the surface of the viral particle.Neutralizing antibodies predominantly bind to the E protein, the preM/Mprotein functions as a chaperone for proper folding of E protein andprevent premature fusion of E protein within acidic compartments alongthe cellular secretory pathway.

Described herein are examples of ZIKV vaccine designs comprising mRNAencoding the both prM/M and E proteins or E protein alone (FIGS. 26A and26B). FIG. 26A depicts mRNA encoding an artificial signal peptide fusedto prM protein fused to E protein. FIG. 2B depicts mRNA encoding anartificial signal peptide fused to E protein.

ZIKV vaccine constructs can encode the prME or E proteins from differentstrains, for example, Brazil_isolate_ZikaSPH2015 or ACD75819_Micronesia,having a signal peptide fused to the N-termini of the antigenicprotein(s). In this example, ZIKV vaccines comprise mRNAs encodingantigenic polypeptides having amino acid sequences of SEQ ID NO: 50-59.The examples are not meant to be limiting.

Example 31 Expression of ZIKV prME Protein in Mammalian Cells Using ZIKVmRNA Vaccine Construct

The ZIKV prME mRNA vaccine construct were tested in mammalian cells(239T cells) for the expression of ZIKV prME protein. 293T cells wereplated in 24-well plates and were transfected with 2 μg of ZIKV prMEmRNA using a Lipofectamine transfection reagent. The cells wereincubated for the expression of the ZIKV prME proteins before they werelysed in an immunoprecipitation buffer containing protease inhibitorcocktails. Reducing agent was not added to the lysis buffer to ensurethat the cellular proteins were in a non-reduced state. Cell lysateswere centrifuged at 8,000×g for 20 mins to collect lysed cellprecipitate. The cell precipitates were then stained with anti ZIKVhuman serum and goat anti-human Alexa Fluor 647. Fluorescence wasdetected as an indication of prME expression (FIG. 28).

The expression of ZIKV prME protein was also detected byfluorescence-activated cell sorting (FACS) using a flow cytometer. 293Fcells (2×10⁶ cells/ml, 30 ml) were transfected with 120 μg PEI, 1 ml of150 mM NaCl, and 60 μg prME mRNA. Transfected cells were incubated for48 hours at 37° C. in a shaker at 130 rpm and under 5% CO₂. The cellswere then washed with PBS buffer containing 2% FBS and fixed in afixation buffer (PBS buffer containing formalin) for 20 minutes at roomtemperature. The fixed cells were permeabilized in a permeabilizationbuffer (PBS+1% Triton X100+1 μl of Golgi plug/ml of cells). Thepermeabilized cells were then stained with anti-ZIKV human serum (1:20dilution) and goat anti-human Alexa Fluor 647 secondary antibody, beforethey were sorted on a flow cytometer. As shown in FIG. 29, FIG. 30A andFIG. 30B, cells transfected with prME mRNA and stained with theanti-ZIKA human serum shifted to higher fluorescent intensity,indicating that prME expressed from the ZIKV mRNA vaccine constructs inthe transfected cells.

Example 32 Expression, Purification and Characterization of Zika VLPs

VLPs were made in HeLa cells and in HEK293t cells and purified via PEGprecipitation or ultracentrifugation, respectively. Cells were culturedin culture media. Prior to transfection, cells were passaged twice invirus growth media+10% FBS to media adaptation.

Cells were seeded the day before transfection into T-175 flask. 100 ugof prME-encoding mRNA was transfected using 100 μg pf lipofectamine asper manufacturer's protocol. 6 hours post transfection, monolayers werewashed twice with 1×PBS and 20 mL of virus growth media was added.Supernatant was collected 24-48 hours post transfection bycentrifugation at 2000×g for 10 mins and 0.22 μm filtration.

For VLP purification via PEG precipitation, VLP's were concentratedusing Biovision PEG precipitation kit as per manufacturer's protocol. Inbrief, supernatant with VLP's was mixed with PEG8000 and incubated at 4°C. for 16 hours. After incubation, mixture was centrifuged at 3000×g for30 mins. Pellet containing concentrated VLP's was collected andsuspended into PBS. VLP's were further buffer exchanged into PBS (1:500)using amicon ultra 100MWCO filter. Purified samples were negativestained (FIG. 32).

Expression of prME from the vaccine mRNA constructs on the invention wasdemonstrated to result in the production of virus like particles (VLPs)that are expected to present to the immune system as identical to Zikavirus particles. FIG. 32 shows negative stain electron micrographs ofsupernatants from HeLa cells transfected with mRNA encoding Zika prME.The virus-like particles (VLPs), purified by PEG precipitation, havehighly uniform size (˜35-40 nm) and morphology. The bumpy appearance ofthe VLP surface appears to reflect mostly immature morphology due toexpression from HeLa cells, which have very low expression of furin, ahost protease that is required for maturation the viral envelope. Uponmaturation, these VLPs will have an exterior structure essentiallyidentical to wild type viral particles, thus eliciting a broad immuneresponse to future Zika virus exposure.

For VLP purification via ultracentrifugation, 293T cells weretransfected with Zika prME mRNA as described herein. Supernatant wascollected 24 hours after changing the media as described herein. (30hours post transfection) VLP's were concentrated using Biovision PEGvirus precipitation kit into 500 μL volume. VLP were further purifiedusing a 10-50% sucrose gradient. Sample layer was seen between 20-30%sucrose layers and collected. VLP's were buffered exchanged into PBS by1:1000 dilution using a 100MWCO amicon ultra filter. VLP's concentratedafter PEG precipitation and ultracentrifuge purified VLP were analyzedon a reducing SDS-PAGE gel for purity (FIG. 33).

Example 33 Immunogenicity Studies

Study A

The instant study was designed to test the immunogenicity in Balb/c miceof candidate ZIKV vaccines comprising a mRNA polynucleotide encodingZIKV prME. Four groups of Balb/c mice (n=5) were immunizedintramuscularly (IM) with 10 μg (n=2) or 2 μg (n=2) of the candidatevaccine. One group of mice was administered PBS intramuscularly as acontrol. All mice were administered an initial dose of vaccine (Groups1-4) or PBS (Group 5) on Day 0, and then the mice in Groups 1 and 3 wereadministered a boost dose on Day 21, while the mice in Group 5 wereadministered PBS on Day 21. All mice were bled on Day 41. See Table 29.Anti-Zika neutralization IgG titer was determined on Day −1, Day 28 andDay 41 (FIG. 33B).

TABLE 29 ZIKV mRNA Vaccine Immunogenicity Study Study design BALB/CImmunization Group Vaccine N Dose Route Prime Boost Endpoint 1 Zika 5 10ug IM Day Day Terminal prME 0 21 bleeds vaccine on Day 41. 2 Zika 5 10ug IM Day NA Anti Zika prME 0 neutralizing vaccine IgG titer. 3 Zika 5 2 ug IM Day Day prME 0 21 vaccine 4 Zika 5  2 ug IM Day NA prME 0vaccine 5 PBS 5 NA IM Day Day 0 21

Day 42 neutralizing titers reached EC50s of 427 for 2 μg and 690 for 10μg. The control serum in this experiment was from naturally infectedimmunocompromised mice (Ifnar1−/−, derived from B/6 lineage) in whichhigh viral loads would be achieved.

Study B

The instant study is designed to test the immunogenicity in mice ofcandidate ZIKV vaccines comprising a mRNA polynucleotide encoding ZIKVpolyprotein. Mice are immunized intravenously (IV), intramuscularly(IM), or intradermally (ID) with candidate vaccines. Up to threeimmunizations are given at 3-week intervals (i.e., at weeks 0, 3, 6, and9), and sera are collected after each immunization until weeks 33-51.Serum antibody titers against ZIKV polyprotein are determined by ELISA.

Example 34 ZIKV Rodent Challenge

Study A

The instant study was designed to test the efficacy in AG129 mice ofcandidate ZIKV vaccines against a lethal challenge using a ZIKV vaccinecomprising mRNA encoding ZIKV prME. Four groups of AG129 mice (n=8) wereimmunized intramuscularly (IM) with 10 μg (n=2) or 2 μg (n=2) of thecandidate vaccine. One group of mice was administered PBSintramuscularly as a control. All mice were administered an initial doseof vaccine (Groups 1-4) or PBS (Group 5) on Day 0, and then the mice inGroups 1 and 3 were administered a boost dose on Day 21, while the micein Group 5 were administered PBS on Day 21. All mice were challengedwith a lethal dose of ZIKV in Day 42. All mice were then monitored forsurvival and weight loss. Anti-Zika neutralization IgG titer wasdetermined on Day −1, Day 28 and Day 41, and viral load was determined 5days post challenge.

TABLE 30 ZIKV In vivo challenge Study design AG129 Immunization Chal-End- Group Vaccine n Dose Route Prime Boost lenge point 1 Zika 8 10 ugIM Day Day Day Monitor prME 0 21 42 for vaccine survival 2 Zika 8 10 ugIM Day NA and prME 0 weight vaccine loss. 3 Zika 8  2 ug IM Day DayViral prME 0 21 load at vaccine Day 5 4 Zika 8  2 ug IM Day NA prME 0vaccine 5 PBS 8 NA IM Day Day 0 21Study B

The instant study is designed to test the efficacy in AG129 mice ofcandidate ZIKV vaccines against a lethal challenge using a ZIKV vaccinecomprising mRNA encoding ZIKV polyprotein. Animals are challenged with alethal dose of the ZIKV. Animals are immunized intravenously (IV),intramuscularly (IM), or intradermally (ID) at week 0 and week 3 withcandidate ZIKV vaccines with and without adjuvant. The animals are thenchallenged with a lethal dose of ZIKV on week 7 via IV, IM or ID.Endpoint is day 13 post infection, death or euthanasia. Animalsdisplaying severe illness as determined by >30% weight loss, extremelethargy or paralysis are euthanized. Body temperature and weight areassessed and recorded daily.

In experiments where a lipid nanoparticle (LNP) formulation is used, theformulation may include a cationic lipid, non-cationic lipid, PEG lipidand structural lipid in the ratios 50:10:1.5:38.5. The cationic lipid isDLin-KC2-DMA or DLin-MC3-DMA (50 mol %), the non-cationic lipid is DSPC(10 mol %), the PEG lipid is PEG-DOMG or PEG-DMG (1.5 mol %) and thestructural lipid is cholesterol (38.5 mol %), for example.

TABLE 31 ZIKV Nucleic Acid Sequences SEQ ID Description Sequence NO:Zika virus ATGAAAAACCCAAAGAAGAAATCCGGAGGATTCCGGATTGTCAATATGCTAAAAC 48strain MR 766 GCGGAGTAGCCCGTGTAAACCCCTTGGGAGGTTTGAAGAGGCTGCCAGCCGGACTpolyprotein TCTGCTGGGTCATGGACCCATCAGAATGGTTTTGGCGATATTAGCCTTTTTGAGAgene, TTCACAGCAATCAAGCCATCACTGGGCCTCATCAACAGATGGGGTACCGTGGGGAcomplete cds AAAAAGAGGCTATGGAAATAATAAAAAAATTTAAGAAAGATCTTGCTGCCATGTTGenBank GAGAATAATCAATGCTAGGAAGGAGAGGAAGAGACGTGGCGCAGACACCAGCATCAccession: GGAATCGTTGGCCTCCTGTTGACTACAGCCATGGCAGCAGAGATCACTAGACGTGDQ859059 GGAGTGCATACTACATGTACTTGGATAGGAGCGATGCAGGGAAGGCCATTTCTTTCGCTACCACATTGGGGGTGAACAAATGCCATGTGCAGATCATGGACCTCGGGCACATGTGTGACGCCACCATGAGCTATGAATGCCCTATGCTGGACGAGGGGGTGGAACCAGATGACGTCGATTGCTGGTGCAACACGACATCAACTTGGGTTGTGTACGGAACCTGTCATCATAAAAAAGGTGAAGCACGGCGATCTAGAAGAGCCGTCACGCTCCCATCTCACTCCACAAGGAAATTGCAAACGCGGTCGCAGACTTGGCTAGAATCAAGAGAATACACAAAGCACCTGATCAAGGTTGAAAATTGGATATTCAGGAACCCTGGTTTTACGCTAGTGGCTGTCGCCATCGCCTGGCTTTTGGGAAGCTCGACGAGCCAAAAAGTCATATACTTGGTCATGATACTGCTGATTGCCCCGGCATACAGTATCAGGTGCATAGGAGTCAGCAATAGAGACTTCGTGGAGGGCATGTCAGGTGGGACCTGGGTTGACGTTGTCCTGGAACATGGAGGCTGCGTCACCGTGATGGCACAGGACAAGCCAACAGTTGACATAGAGCTGGTCACAACAACGGTTAGTAACATGGCCGAGGTGAGATCCTATTGTTACGAGGCATCAATATCGGACATGGCTTCGGACAGTCGCTGCCCAACACAAGGTGAAGCCTACCTTGACAAGCAATCAGACACTCAATATGTTTGCAAAAGAACATTGGTGGACAGAGGTTGGGGAAATGGGTGTGGACTCTTTGGCAAAGGGAGTTTGGTGACATGTGCTAAGTTCACGTGCTCCAAGAAGATGACTGGGAAGAGCATTCAGCCGGAGAACCTGGAGTATCGGATAATGCTATCAGTGCATGGCTCCCAGCACAGTGGGATGATTGTTAATGATGAAAACAGAGCGAAGGTCGAGGTTACGCCCAATTCACCAAGAGCAGAAGCAACCCTGGGAGGCTTTGGAAGCTTAGGACTTGATTGTGAACCAAGGACAGGCCTTGACTTTTCAGATCTGTATTACCTAACCATGAATAACAAGCATTGGTTGGTGCACAAAGAGTGGTTTCATGACATCCCATTGCCCTGGCATGCTGGGGCAGACACTGGAACTCCACATTGGAACAACAAGGAGGCATTAGTGGAATTCAAGGACGCCCACGCCAAGAGGCAAACCGTCGTGGTTTTGGGGAGCCAGGAAGGAGCCGTCCACACGGCTCTTGCTGGAGCTCTAGAGGCTGAGATGGATGGTGCAAAGGGAAGGCTATTCTCTGGCCACTTGAAATGTCGCTTAAAAATGGACAAGCTTAGATTGAAGGGCGTGTCATATTCCTTGTGCACCGCGGCATTCACATTCACCAAGGTCCCGGCTGAAACACTACATGGAACAGTCACAGTGGAGGTGCAGTATGCAGGGACAGATGGACCCTGCAAGGTCCCAGCCCAGATGGCGGTGGACATGCAGACCTTGACCCCAGTCGGAAGGCTGATAACCGCCAACCCCGTGATTACTGAAAGCACTGAGAATTCAAAGATGATGTTGGAGCTCGACCCACCATTTGGGGATTCTTACATTGTCATAGGAGTTGGGGATAAGAAAATCACCCATCACTGGCATAGGAGTGGCAGCACCATTGGAAAAGCATTTGAAGCCACTGTGAGAGGCGCTAAGAGAATGGCAGTCCTGGGGGACACAGCTTGGGACTTTGGATCAGTCGGAGGTGTGTTTAACTCATTGGGCAAGGGCATTCATCAGATTTTTGGAGCAGCTTTCAAATCACTGTTTGGAGGAATGTCCTGGTTCTCACAGATCCTCATAGGCACTCTGCTGGTGTGGTTAGGTCTGAACACAAAGAATGGGTCTATCTCCCTCACATGCTTAGCCCTGGGGGGAGTGATGATCTTCCTCTCCACGGCTGTTTCTGCTGACGTGGGGTGCTCGGTGGACTTCTCAAAAAAAGAAACGAGATGTGGCACGGGGGTGTTCGTCTACAATGACGTTGAAGCCTGGAGGGACCGGTACAAGTACCATCCTGACTCCCCTCGTAGACTGGCAGCAGCCGTTAAGCAAGCTTGGGAAGAGGGGATTTGTGGGATCTCCTCTGTTTCTAGAATGGAAAACATAATGTGGAAATCAGTGGAAGGAGAGCTCAATGCAATCCTAGAGGAGAATGGAGTCCAACTGACAGTTGTTGTGGGATCTGTAAAAAACCCCATGTGGAGAGGCCCACAAAGATTGCCAGTGCCTGTGAATGAGCTGCCCCATGGCTGGAAAGCCTGGGGGAAATCGTACTTTGTTAGGGCGGCAAAGACCAACAACAGTTTTGTTGTCGACGGTGACACATTGAAGGAATGTCCGCTCAAGCACAGAGCATGGAACAGCTTCCTCGTGGAGGATCACGGGTTTGGGGTCTTCCACACCAGTGTTTGGCTTAAGGTTAGAGAAGATTACTCACTGGAGTGTGACCCAGCCGTCATAGGAACAGCTGTTAAGGGAAAGGAGGCCGCGCACAGTGATCTAGGCTATTGGATTGAAAGTGAAAAGAATGACACATGGAGGCTGAAGAGGGCTCATTTGATTGAGATGAAAACATGTGAGTGGCCAAAGTCTCACACACTGTGGACAGATGGAGTGGAAGAAAGTGATCTGATCATACCCAAGTCTTTAGCTGGTCCACTCAGCCACCACAACACCAGAGAGGGTTACAGAACTCAAGTGAAAGGGCCATGGCATAGTGAGGAGCTTGAAATCCGATTTGAGGAATGTCCAGGTACCAAGGTTCATGTGGAGGAGACATGCGGAACGAGAGGACCATCTCTGAGATCAACCACTGCAAGCGGAAGGGTCATTGAGGAATGGTGCTGTAGGGAATGCACAATGCCCCCACTATCGTTCCGAGCAAAAGATGGCTGCTGGTATGGAATGGAGATAAGGCCTAGGAAAGAACCAGAGAGCAACTTAGTGAGGTCAATGGTGACAGCGGGATCAACCGATCATATGGATCATTTTTCTCTTGGAGTGCTTGTGATTCTACTCATGGTGCAGGAAGGGTTGAAGAAGAGAATGACCACAAAGATCATCATGAGCACATCAATGGCAGTGCTGGTGGCCATGATCTTGGGAGGATTCTCAATGAGTGACCTGGCTAAGCTTGTGATCCTGATGGGGGCCACTTTCGCAGAAATGAACACTGGAGGAGACGTAGCTCACTTGGCATTAGTAGCGGCATTTAAAGTCAGACCAGCCTTGCTGGTCTCATTTATCTTCAGAGCCAACTGGACACCTCGTGAGAGCATGCTGCTAGCCTTGGCTTCGTGTCTTCTGCAAACTGCGATCTCCGCTCTTGAAGGCGACTTGATGGTCCTCGTTAATGGATTTGCTTTGGCCTGGTTGGCAATACGTGCAATGGCCGTGCCACGCACTGACAACATCGCTCTAGCAATTCTGGCTGCTCTAACACCACTAGCCCGAGGCACACTGCTCGTGGCATGGAGAGCGGGCCTCGCCACTTGTGGAGGGTTCATGCTCCTCTCCCTGAAAGGGAAAGGTAGTGTGAAGAAGAACCTGCCATTCGTCGCGGCCTTGGGATTGACCGCTGTGAGAATAGTGGACCCCATTAATGTGGTGGGACTACTGTTACTCACAAGGAGTGGGAAGCGGAGCTGGCCCCCTAGTGAAGTGCTCACTGCTGTCGGCCTGATATGTGCATTGGCCGGAGGGTTTGCCAAGGCAGACATAGAGATGGCTGGGCCCATGGCGGCAGTGGGCCTGCTAATTGTCAGTTATGTGGTCTCGGGAAAGAGTGTAGATATGTACATTGAAAGAGCAGGTGACATCACATGGGAGAAAGACGCGGAAGTCACTGGAAACAGTCCTCGGCTTGACGTGGCACTAGATGAGAGTGGTGATTTCTCTCTGGTGGAGGAAGATGGTCCACCCATGAGAGAGATCATACTTAAGGTGGTCTTGATGGCCATCTGTGGCATGAACCCAATAGCCATACCTTTTGCTGCAGGAGCGTGGTATGTGTATGTGAAGACTGGGAAAAGGAGTGGTGCCCTCTGGGACGTGCCTGCTCCGAAAGAAGTGAAAAAAGGAGAGACCACAGATGGAGTGTACAGAGTGATGACTCGCAGACTGCTGGGTTCAACACAAGTTGGAGTGGGAGTCATGCAGGAGGGAGTCTTCCACACCATGTGGCACGTCACAAAAGGGGCCGCATTGAGGAGCGGTGAAGGGAGACTTGATCCATACTGGGGGGATGTCAAGCAGGACTTGGTGTCATATTGTGGGCCTTGGAAGCTGGACGCAGCTTGGGACGGAGTTAGTGAGGTGCAGCTTCTGGCCGTACCCCCTGGAGAGAGAGCCAGAAACATTCAGACTCTGCCTGGAATATTTAAGACAAAGGATGGGGACATCGGAGCAGTTGCTTTGGACTATCCTGCAGGAACCTCAGGATCTCCGATCCTAGACAAATGCGGGAGAGTGATAGGACTCTATGGCAATGGGGTTGTGATCAAGAACGGAAGCTATGTTAGTGCTATAACCCAGGGAAAGAGGGAGGAGGAGACTCCGGTTGAGTGTTTTGAACCCTCGATGCTGAAGAAGAAGCAGCTAACTGTCCTGGACCTGCATCCAGGGGCTGGGAAAACCAGGAGAGTTCTTCCTGAAATAGTCCGTGAAGCTATAAAGAAGAGACTCCGCACGGTGATCTTGGCACCAACCAGGGTCGTCGCTGCTGAGATGGAGGAAGCCCTGAGAGGACTTCCGGTGCGTTACATGACAACAGCAGTCAAGGTCACCCATTCTGGGACAGAAATCGTTGATTTGATGTGCCATGCCACCTTCACTTCACGCCTACTACAACCCATTAGAGTCCCTAATTACAACCTCTACATCATGGATGAAGCCCATTTCACAGACCCCTCAAGCATAGCTGCAAGAGGATATATATCAACAAGGGTTGAGATGGGCGAGGCAGCAGCCATCTTTATGACTGCCACACCACCAGGAACCCGCGATGCGTTTCCAGATTCCAACTCACCAATCATGGACACAGAAGTGGAAGTCCCAGAGAGAGCCTGGAGCTCAGGCTTTGATTGGGTGACGGACCATTCTGGGAAAACAGTTTGGTTCGTTCCAAGCGTGAGGAATGGAAATGAAATCGCAGCCTGTCTGACAAAGGCTGGAAAGCGGGTTATACAGCTTAGTAGGAAAACTTTTGAGACAGAGTTTCAGAAAACAAAAAATCAAGAGTGGGACTTTGTCATAACAACTGACATCTCAGAGATGGGTGCCAACTTCAAGGCTGACCGGGTTATAGATTCCAGGAGATGCCTAAAGCCAGTTATACTTGATGGTGAGAGAGTCATCTTGGCTGGGCCCATGCCTGTCACGCATGCTAGCGCTGCTCAGAGGAGAGGACGTATAGGCAGGAACCCCAACAAGCCTGGAGATGAGTACATGTATGGAGGTGGGTGTGCGGAGACTGATGAAGACCATGCACATTGGCTTGAAGCAAGAATGCTTCTTGACAACATTTACCTCCAGGATGGCCTCATAGCCTCGCTCTATCGACCTGAGGCCGACAAGGTAGCCGCCATTGAGGGAGAGTTTAAGCTGAGGACAGAGCAAAGGAAGACCTTTGTGGAACTCATGAAGAGAGGAGATCTTCCCGTTTGGTTGGCCTACCAGGTTGCATCTGCCGGAATAACTTATACAGACAGAAGATGGTGTTTTGATGGCACAACCAACAACACCATAATGGAAGACAGTGTACCAGCAGAGGTGTGGACCAAGTATGGAGAGAAGAGAGTGCTCAAACCAAGATGGATGGACGCCAGGGTCTGCTCAGATCATGCGGCCCTGAAGTCGTTCAAAGAATTCGCCGCTGGGAAAAGAGGAGCGGCTTTGGGAGTAATGGAGGCCCTGGGAACATTACCAGGACACATGACAGAGAGGTTTCAGGAAGCCATTGATAACCTCGCTGTGCTCATGCGAGCAGAGACTGGAAGCAGGCCCTACAAGGCAGCGGCAGCCCAATTGCCGGAGACCCTAGAGACCATCATGCTTTTAGGCCTGCTGGGAACAGTATCGCTGGGGATCTTTTTTGTCTTGATGAGGAACAAGGGCATCGGGAAGATGGGCTTTGAAATGGTAACCCTTGGGGCCAGCGCATGGCTCATGTGGCTCTCAGAAATCGAACCAGCCAGAATTGCATGTGTCCTTATTGTTGTGTTTTTATTACTGGTGGTGCTAATACCAGAGCCAGAGAAGCAAAGATCCCCCCAGGACAATCAGATGGCAATCATTATTATGGTGGCAGTGGGCCTTTTGGGGTTGATAACTGCAAATGAACTTGGATGGCTGGAGAGAACAAAAAATGACATAGCTCATCTGATGGGAAAGAGAGAAGAGGGAACAACCGTGGGATTCTCAATGGACATCGATCTGCGACCAGCCTCCGCATGGGCTATTTATGCCGCATTGACAACCCTCATCACCCCAGCCGTCCAGCACGCGGTAACTACCTCGTACAACAACTACTCCTTAATGGCGATGGCCACACAAGCTGGAGTGCTGTTTGGCATGGGCAAAGGGATGCCATTTTATGCATGGGACTTAGGAGTCCCGTTGCTAATGATGGGCTGCTACTCACAACTAACACCCCTGACCCTGATAGTAGCCATCATTTTGCTTGTGGCACATTACATGTACTTGATCCCAGGCCTACAGGCAGCAGCAGCACGCGCTGCCCAGAAGAGAACAGCAGCCGGCATCATGAAGAATCCCGTTGTGGATGGAATAGTGGTAACTGACATTGACACAATGACAATTGACCCCCAAGTGGAGAAGAAGATGGGACAAGTGCTACTTATAGCAGTGGCTGTCTCCAGTGCTGTGTTGCTGCGGACCGCTTGGGGATGGGGGGAGGCTGGAGCTTTGATCACAGCAGCAACTTCCACCCTGTGGGAAGGCTCCCCAAACAAATACTGGAACTCCTCCACAGCCACCTCACTGTGCAACATCTTCAGAGGAAGTTACTTGGCAGGAGCTTCCCTTATTTACACAGTGACAAGAAATGCCGGCCTGGTTAAGAGACGTGGAGGTGGAACGGGAGAAACTCTGGGAGAGAAGTGGAAAGCCCGCCTGAATCAGATGTCGGCCTTGGAGTTCTACTCTTACAAAAAGTCAGGCATCACTGAAGTATGTAGAGAGGAGGCTCGCCGCGCCCTCAAGGATGGAGTGGCCACAGGAGGACATGCTGTATCCCGAGGAAGCGCAAAACTCAGATGGTTGGTGGAGAGAGGATATCTGCAGCCCTATGGAAAGGTTGTTGATCTCGGATGCGGCAGAGGGGGCTGGAGTTATTATGCCGCCACCATCCGCAAAGTGCAGGAGGTGAGAGGATACACAAAGGGAGGTCCCGGTCATGAAGAGCCCATGCTGGTGCAAAGCTATGGGTGGAACATAATTCGTCTCAAGAGTGGAGTGGACGTCTTCCACATGGCGGCTGAGTCGTGTGACACTTTGCTGTGTGACATAGGTGAGTCATCATCCAGTCCTGAAGTGGAGGAGACGCGAACACTCAGAGTGCTCTCCATGGTGGGGGACTGGCTTGAGAAGAGACCAGGGGCCTTCTGCATAAAGGTGTTATGCCCATACACCAGCACCATGATGGAGACCATGGAGCGACTGCAACGTAGGTATGGGGGAGGACTAGTCAGAGTGCCACTGTCCCGCAATTCTACACATGAGATGTATTGGGTCTCTGGAGCAAAAAGTAACATCATAAAAAGTGTGTCCACCACAAGTCAGCTCCTCCTGGGACGCATGGATGGGCCCAGGAGGCCAGTGAAGTATGAGGAGGATGTGAACCTCGGCTCAGGCACACGAGCTGTGGCAAGCTGTGCTGAGGCTCCCAACATGAAGGTCATTGGTAGGCGCATTGAGAGAATCCGTAGTGAACATGCAGAAACATGGTTCTTTGATGAAAACCATCCATACAGGACATGGGCCTACCACGGGAGCTACGAAGCCCCCACGCAAGGGTCAGCATCTTCCCTCGTGAATGGGGTTGTTAGACTCCTGTCAAAGCCCTGGGATGTGGTGACTGGAGTTACAGGAATAGCTATGACTGACACCACACCGTACGGCCAACAAAGAGTCTTCAAAGAAAAAGTGGACACCAGGGTGCCAGACCCTCAAGAAGGTACTCGCCAGGTAATGAACATGGTCGCTTCCTGGCTGTGGAAGGAGCTGGGAAAACGTAAGCGGCCACGTGTCTGCACCAAAGAAGAGTTCATCAACAAGGTGCGCAGCAATGCAGCACTGGGAGCAATATTTGAAGAGGAAAAAGAATGGAAGACGGCTGTGGAAGCTGTGAATGATCCAAGGTTTTGGGCCCTAGTGGATAAGGAAAGAGAACACCACCTGAGAGGAGAGTGCCATAGTTGTGTGTACAACATGATGGGAAAAAGAGAAAAGAAGCAAGGGGAATTCGGGAAAGCAAAAGGCAGTCGCGCCATCTGGTACATGTGGTTGGGAGCCAGATTCTTGGAGTTTGAAGCCCTTGGATTCTTGAACGAGGACCATTGGATGGGAAGAGAAAACTCAGGAGGTGGTGTCGAAGGGTTGGGACTGCAAAGACTTGGATACGTTCTAGAAGAAATGAGCCGGGCACCAGGAGGAAAGATGTATGCAGATGACACCGCTGGCTGGGACACCCGCATTAGCAAGTTTGATTTGGAGAATGAAGCCTTGATTACTAACCAAATGGATGAAGGGCACAGAACTCTGGCGTTGGCCGTGATTAAGTACACATACCAAAACAAAGTGGTGAAGGTCCTCAGACCAGCTGAAGGAGGAAAAACAGTCATGGACATCATTTCAAGACAAGACCAGAGGGGGAGCGGACAAGTTGTCACTTATGCTCTCAACACATTTACCAACTTGGTGGTGCAGCTCATCCGGAACATGGAGGCTGAGGAAGTGTTAGAGATGCAAGACTTATGGCTGTTGAGGAAGCCAGAGAAAGTAACCAGATGGCTGCAGAGTAGCGGATGGGACAGACTCAAACGAATGGCAGTCAGTGGTGATGACTGTGTTGTAAAGCCAATTGATGACAGGTTTGCACACGCCCTCAGGTTCTTGAATGATATGGGGAAAGTTAGGAAAGACACACAGGAATGGAAACCCTCAACTGGATGGAGCAACTGGGAAGAAGTCCCGTTCTGCTCCCACCACTTTAACAAGCTGCACCTCAAAGACGGGAGATCCATTGTGGTCCCTTGCCGCCACCAAGATGAACTGATTGGCCGGGCTCGCGTTTCGCCGGGGGCAGGATGGAGCATCCGGGAGACTGCCTGTCTTGCAAAATCATATGCACAGATGTGGCAGCTTCTTTATTTCCACAGAAGAGACCTCCGACTGATGGCCAATGCCATTTGCTCGGCCGTGCCAGTTGACTGGGTCCCAACTGGGAGAACTACCTGGTCAATCCATGGAAAGGGAGAATGGATGACTACTGAGGACATGCTCATGGTGTGGAATAGAGTGTGGATTGAGGAGAATGATCACATGGAGGACAAGACCCCTGTAACAAAATGGACAGACATTCCCTATTTGGGAAAAAGGGAGGACTTATGGTGTGGATCCCTTATAGGACACAGACCTCGCACCACTTGGGCTGAGAACATCAAAGACACAGTCAGCATGGTGCGCAGAATCATAGGTGATGAAGAAAAGTACATGGACTACCTATCCACTCAAGTTCGCTACTTGGGTGAGGAAGGGTCTACACCTGGAGTGCTGTAA IgE HC signalTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG 49 peptide_prM-EAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGACTGGACCTGGATC #1CTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCGTGGAAGTGACCAGACGGG(Brazil_isolate_ZikaSPH2015,GCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGCCATCAGCTT Sequence,TCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTGGGCCAC NT (5′ UTR,ATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGGGCGTGGAAC ORF, 3′ UTR)CCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACAGCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCTCCCAGCACAGCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGATCCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACAGCCTGGGAAAGGGCATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCGGCGTGCTGATCTTTCTGAGCACAGCCGTGTCCGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC IgE HC signalATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCG 50 peptide_prM-ETGGAAGTGACCAGACGGGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGC #1CGGCGAGGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAG(Brazil_isolate_ZikaSPH2015),ATCATGGACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGC ORFTGGACGAGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCAC Sequence, NTCTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACAGCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCTCCCAGCACAGCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGATCCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACAGCCTGGGAAAGGGCATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCGGCGTGCTGATCTTTCTGAGCACAGCCGTGTCCGCC IgE HC signalG*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAC 51 peptide_prM-ETGGACCTGGATCCTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCGTGGAAG #1TGACCAGACGGGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGA(Brazil_isolate_ZikaSPH2015),GGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATG mRNAGACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACG SequenceAGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGT (T100 tail)GGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACAGCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCTCCCAGCACAGCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGATCCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACAGCCTGGGAAAGGGCATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCGGCGTGCTGATCTTTCTGAGCACAGCCGTGTCCGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCTAG IgE HC signalTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG 52 peptide_prM-EAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGACTGGACCTGGATC #2CTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCACCAGAAGAGGCAGCGCCT(Brazil_isolate_ZikaSPH2015),ACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGCCATCAGCTTTCCAACCAC Sequence,CCTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTGGGCCACATGTGCGAC NT (5′ UTR,GCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGGGCGTGGAACCCGACGATG ORF, 3′ UTR)TGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCTCCCAGCACAGCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGATCCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACAGCCTGGGAAAGGGCATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCGGCGTGCTGATCTTTCTGAGCACAGCCGTGTCCGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC IgE HC signalATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCA 53 peptide_prM-ECCAGAAGAGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGC #2CATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGAC(Brazil_isolate_ZikaSPH2015),CTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGG ORFGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGT Sequence, NTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCTCCCAGCACAGCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGATCCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACAGCCTGGGAAAGGGCATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCGGCGTGCTGATCTTTCTGAGCACAGCCGTGTCCGCC IgE HC signalG*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAC 54 peptide_prM-ETGGACCTGGATCCTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCACCAGAA #2GAGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGCCATCAG(Brazil_isolate_ZikaSPH2015),CTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTGGGC mRNACACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGGGCGTGG SequenceAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGG (T100 tail)CACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCTCCCAGCACAGCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGATCCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACAGCCTGGGAAAGGGCATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCGGCGTGCTGATCTTTCTGAGCACAGCCGTGTCCGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCTAG HuIgG_(k )signalTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG 55 peptide_prMEAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAAACCCCTGCCCAG #1CTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCACCGGCGTGGAAGTGACCA(Brazil_isolate_ZikaSPH2015),GAAGAGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGCCAT Sequence,CAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTG NT (5′ UTR,GGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGGGCG ORF, 3′ UTR)TGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACCGCAGCGGCAGCACAATCGGCAAGGCCTTTGAAGCCACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGATCTTTCTGAGCACCGCCGTGTCTGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC HuIgG_(k )signalATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCA 56 peptide_prMECCGGCGTGGAAGTGACCAGAAGAGGCAGCGCCTACTACATGTACCTGGACAGAAG #1CGACGCCGGCGAGGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTAC(Brazil_isolate_ZikaSPH2015),ATCCAGATCATGGACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCC ORFCCATGCTGGACGAGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCAC Sequence, NTCAGCACCTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACCGCAGCGGCAGCACAATCGGCAAGGCCTTTGAAGCCACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGATCTTTCTGAGCACCGCCGTGTCTGCC HuIgG_(k )signalG*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAA 57 peptide_prMEACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCACCGGCG #1TGGAAGTGACCAGAAGAGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGC(Brazil_isolate_ZikaSPH2015),CGGCGAGGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAG mRNAATCATGGACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGC SequenceTGGACGAGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCAC (T100 tail)CTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACCGCAGCGGCAGCACAATCGGCAAGGCCTTTGAAGCCACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGATCTTTCTGAGCACCGCCGTGTCTGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCTAG HuIgG_(k )signalTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG 58 peptide_prMEAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAAACCCCTGCCCAG #2CTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCACCGGCACCAGAAGAGGCA(Brazil_isolate_ZikaSPH2015),GCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGCCATCAGCTTTCC Sequence,AACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGACCTGGGCCACATG NT (5′ UTR,TGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGGGCGTGGAACCCG ORF, 3′ UTR)ACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACCGCAGCGGCAGCACAATCGGCAAGGCCTTTGAAGCCACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGATCTTTCTGAGCACCGCCGTGTCTGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC HuIgG_(k )signalATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCA 59 peptide_prMECCGGCACCAGAAGAGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGG #2CGAGGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATC(Brazil_isolate_ZikaSPH2015),ATGGACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGG ORFACGAGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTG Sequence, NTGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACCGCAGCGGCAGCACAATCGGCAAGGCCTTTGAAGCCACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGATCTTTCTGAGCACCGCCGTGTCTGCC HuIgG_(k )signalG*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAA 60 peptide_prMEACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGATACCACCGGCA #2CCAGAAGAGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGCCGGCGAGGC(Brazil_isolate_ZikaSPH2015),CATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATGGAC mRNACTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACGAGG SequenceGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGTGGT (T100 tail)GTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACTCCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACCGCAGCGGCAGCACAATCGGCAAGGCCTTTGAAGCCACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGATCTTTCTGAGCACCGCCGTGTCTGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCTAG HuIgG_(k )signalTCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAG 61 peptide_EAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAAACCCCTGCCCAG(Brazil_isolate_ZikaSPH2015),CTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCATCAGATGCATCG Sequence,GCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGT NT (5′ UTR,GGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTG ORF, 3′ UTR)GACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGACAAGCAGAGCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGAGCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGGCTGATCACAGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGAAGCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGATCTTTCTGAGCACCGCCGTGTCTGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC HuIgG_(k )signalATGGAAACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCA 62 peptide_ECCGGCATCAGATGCATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGG(Brazil_isolate_ZikaSPH2015),CGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCC ORFCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGG Sequence, NTCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGACAAGCAGAGCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGAGCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGGCTGATCACAGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGAAGCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGATCTTTCTGAGCACCGCCGTGTCTGCC HuIgG_(k )signalG*GGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAA 63 peptide_EACCCCTGCCCAGCTGCTGTTCCTGCTGCTGCTGTGGCTGCCTGACACCACCGGCA(Brazil_isolate_ZikaSPH2015),TCAGATGCATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCAC mRNAATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGAT SequenceAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAG (T100 tail)TGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGACAAGCAGAGCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGAGCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGGCTGATCACAGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGAAGCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGAGGCGTGCTGATCTTTCTGAGCACCGCCGTGTCTGCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATCTAG Zika_RIO-ATGCTGGGCAGCAACAGCGGCCAGAGAGTGGTGTTCACCATCCTGCTGCTGCTGG 64 U1_JEVspTGGCCCCTGCCTACAGCGCCGAAGTGACAAGAAGAGGCAGCGCCTACTACATGTA Zika PRMECCTGGACCGGAACGATGCCGGCGAGGCCATCAGCTTTCCAACCACCCTGGGCATG StrainAACAAGTGCTACATCCAGATCATGGACCTGGGCCACATGTGCGACGCCACCATGA ascension id:GCTACGAGTGCCCCATGCTGGACGAGGGCGTGGAACCCGACGATGTGGACTGCTG ANG09399 withGTGCAATACCACCAGCACCTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGC JEV PRMGAAGCCAGACGGTCCAGACGGGCCGTGACACTGCCTAGCCACAGCACCAGAAAGC signalTGCAGACCCGGTCCCAGACCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGAT sequenceCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCTGCCGCTGCT (optimized)ATTGCTTGGCTGCTGGGCTCTAGCACCAGCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCAGCCTACTCCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGACAAGCCCACCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGACAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGACAGGGGCTGGGGCAATGGCTGTGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCTCCCAGCACAGCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAACACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTGGAACAACAAAGAGGCCCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGAGCAGCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGGCTGATCACAGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGAAGCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACAGCCTGGGAAAGGGCATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTGATGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGATGTGCCTGGCTCTGGGCGGCGTGCTGATCTTTCTGAGCACAGCCGTGTCCGCC Zika_RIO-ATGAAGTGCCTGCTGTACCTGGCCTTCCTGTTCATCGGCGTGAACTGCGCCGAAG 65 U1¬_VSVgSpTGACCAGAAGAGGCAGCGCCTACTACATGTACCTGGACCGGAACGATGCCGGCGA Zika PRMEGGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAGATCATG StrainGACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGCTGGACG ascension id:AGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCACCTGGGT ANG09399 withGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGACGGGCC VSV g proteinGTGACACTGCCTAGCCACAGCACCAGAAAGCTGCAGACCCGGTCCCAGACCTGGC signalTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCG sequenceGAACCCCGGCTTTGCCCTGGCCGCTGCTGCTATTGCTTGGCTGCTGGGCAGCAGC (optimized)ACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGACAAGCCCACCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGACAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGTGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCAGCCAGCACTCCGGCATGATCGTGAACGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCCGGCGCTGATACAGGCACACCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCAGCGGCCACCTGAAGTGCAGACTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCAGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGAAGCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGAGATACCGCCTGGGACTTTGGCTCTGTGGGCGGAGCCCTGAACTCTCTGGGCAAGGGAATCCACCAGATCTTCGGAGCCGCCTTTAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTGATGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGATGTGCCTGGCTCTGGGAGGCGTGCTGATCTTCCTGAGCACAGCCGTGTCTGCC ZIKA_PRME_DSP_N154AATGGACTGGACCTGGATCCTGTTCCTGGTGGCCGCTGCCACAAGAGTGCACAGCG 66 Zika PRMETGGAAGTGACCAGACGGGGCAGCGCCTACTACATGTACCTGGACAGAAGCGACGC StrainCGGCGAGGCCATCAGCTTTCCAACCACCCTGGGCATGAACAAGTGCTACATCCAG ascension id:ATCATGGACCTGGGCCACATGTGCGACGCCACCATGAGCTACGAGTGCCCCATGC ACD75819 withTGGACGAGGGCGTGGAACCCGACGATGTGGACTGCTGGTGCAACACCACCAGCAC IgE signalCTGGGTGGTGTACGGCACCTGTCACCACAAGAAGGGCGAAGCCAGACGGTCCAGA peptideCGGGCCGTGACACTGCCTAGCCACAGCACCAGAAAGCTGCAGACCCGGTCCCAGA (optimized)CCTGGCTGGAAAGCAGAGAGTACACCAAGCACCTGATCCGGGTGGAAAACTGGATCTTCCGGAACCCCGGCTTTGCCCTGGCTGCCGCTGCTATTGCTTGGCTGCTGGGCAGCAGCACCTCCCAGAAAGTGATCTACCTCGTGATGATCCTGCTGATCGCCCCTGCCTACAGCATCCGGTGTATCGGCGTGTCCAACCGGGACTTCGTGGAAGGCATGAGCGGCGGCACATGGGTGGACGTGGTGCTGGAACATGGCGGCTGCGTGACAGTGATGGCCCAGGATAAGCCCGCCGTGGACATCGAGCTCGTGACCACCACCGTGTCCAATATGGCCGAAGTGCGGAGCTACTGCTACGAGGCCAGCATCAGCGACATGGCCAGCGACAGCAGATGCCCTACACAGGGCGAGGCCTACCTGGATAAGCAGTCCGACACCCAGTACGTGTGCAAGCGGACCCTGGTGGATAGAGGCTGGGGCAATGGCTGCGGCCTGTTTGGCAAGGGCAGCCTCGTGACCTGCGCCAAGTTCGCCTGCAGCAAGAAGATGACCGGCAAGAGCATCCAGCCCGAGAACCTGGAATACCGGATCATGCTGAGCGTGCACGGCTCCCAGCACAGCGGCATGATCGTGGCCGACACCGGCCACGAGACAGACGAGAACCGGGCCAAGGTGGAAATCACCCCCAACAGCCCTAGAGCCGAGGCCACACTGGGCGGCTTTGGATCTCTGGGCCTGGACTGCGAGCCTAGAACCGGCCTGGATTTCAGCGACCTGTACTACCTGACCATGAACAACAAGCACTGGCTGGTGCACAAAGAGTGGTTCCACGACATCCCCCTGCCCTGGCATGCTGGCGCTGATACAGGCACCCCCCACTGGAACAACAAAGAGGCTCTGGTGGAATTCAAGGACGCCCACGCCAAGCGGCAGACCGTGGTGGTGCTGGGATCTCAGGAAGGCGCCGTGCATACAGCTCTGGCTGGCGCCCTGGAAGCCGAAATGGATGGCGCCAAAGGCAGACTGTCCTCCGGCCACCTGAAGTGCCGGCTGAAGATGGACAAGCTGCGGCTGAAGGGCGTGTCCTACAGCCTGTGTACCGCCGCCTTCACCTTCACCAAGATCCCCGCCGAGACACTGCACGGCACCGTGACTGTGGAAGTGCAGTACGCCGGCACCGACGGCCCTTGTAAAGTGCCTGCTCAGATGGCCGTGGATATGCAGACCCTGACCCCCGTGGGCAGACTGATCACCGCCAACCCTGTGATCACCGAGAGCACCGAGAACAGCAAGATGATGCTGGAACTGGACCCCCCCTTCGGCGACTCCTACATCGTGATCGGCGTGGGAGAGAAGAAGATCACCCACCACTGGCACAGATCCGGCAGCACCATCGGCAAGGCCTTTGAGGCTACAGTGCGGGGAGCCAAGAGAATGGCCGTGCTGGGCGATACCGCCTGGGATTTTGGCTCTGTGGGCGGAGCCCTGAACAGCCTGGGAAAGGGCATCCACCAGATCTTCGGCGCTGCCTTCAAGAGCCTGTTCGGCGGCATGAGCTGGTTCAGCCAGATCCTGATCGGCACCCTGCTCGTGTGGCTGGGCCTGAACACCAAGAACGGCAGCATCTCCCTGACCTGCCTGGCTCTGGGCGGCGTGCTGATCTTTCTGAGCACAGCCGTGTCCGCC

TABLE 32 ZIKV Amino Acid Sequences SEQ ID Description Sequence NO:FSM|ACD75819 MKNPKEEIRRIRIVNMLKRGVARVSPFGGLKRLPAGLLLGHGPIRMVLAI 67polyprotein LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKEKKRRGTDTSVGIVGLLLTTAMAVEVTRRGSAYYMYLDRSDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA MR_766|ABI54475MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI 68LAFLRFTAIKPSLGLINRWGTVGKKEAMEIIKKFKKDLAAMLRIINARKERKRRGADTSIGIVGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFTLVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA SM_6_V_1|ABI54480MKNPKRAGSSRLVNMLRRGAARVIPPGGGLKRLPVGLLLGRGPIKMILAI 69LAFLRFTAIKPSTGLINRWGKVGKKEAIKILTKFKADVGTMLRIINNRKTKKRGVETGIVFLALLVSIVAVEVTKKGDTYYMFADKKDAGKVVTFETESGPNRCSIQAMDIGHMCPATMSYECPVLEPQYEPEDVDCWCNSTAAWIVYGTCTHKTTGETRRSRRSITLPSHASQKLETRSSTWLESREYSKYLIKVENWILRNPGYALVAAVIGWTLGSSRSQKIIFVTLLMLVAPAYSIRCIGIGNRDFIEGMSGGTWVDIVLEHGGCVTVMSNDKPTLDFELVTTTASNMAEVRSYCYEANISEMASDSRCPTQGEAYLDKMADSQFVCKRGYVDRGWGNGCGLFGKGSIVTCAKFTCVKKLTGKSIQPENLEYRVLVSVHASQHGGMINNDTNHQHDKENRARIDITASAPRVEVELGSFGSFSMECEPRSGLNFGDLYYLTMNNKHWLVNRDWFHDLSLPWHTGATSNNHHWNNKEALVEFREAHAKKQTAVVLGSQEGAVHAALAGALEAESDGHKATIYSGHLKCRLKLDKLRLKGMSYALCTGAFTFARTPSETIHGTATVELQYAGEDGPCKVPIVITSDTNSMASTGRLITANPVVTESGANSKMMVEIDPPFGDSYIIVGTGTTKITHHWHRAGSSIGRAFEATMRGAKRMAVLGDTAWDFGSVGGMFNSVGKFVHQVFGSAFKALFGGMSWFTQLLIGFLLIWMGLNARGGTVAMSFMGIGAMLIFLATSVSG MR_766|AAV34151MKNPKEEIRRIRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI 70LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKERKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIGYETDEDRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKIPVQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA MR_766|YP_002790881MKNPKEEIRRIRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI 71LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKERKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCKKMTGKSIQPENLEYRIMLSVHGSQHSGMIGYETDEDRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKIPVQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA ARB7701|AHF49785MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI 72LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKERKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATNLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGVHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA ARB15076|AHF49784MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI 73LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKERKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATNLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA ARB13565|AHF49783MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI 74LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKERKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATNLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGVHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA ArB1362|AHL43500MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI 75LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKERKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSSTSQKVIYLIMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDXXXXXXXNRAEVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA ArD7117|AHL43501MKNPKKRSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI 76LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKERKRRGADTSIGIVGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCQHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAVCTAAKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA ArD157995|AHL43503MKNPKKKSGRFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI 77LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKERKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGETRRSRRSVSLRYHYTRKLQTRSQTWLESREYKKHLIMVENWIFRNPGFAIVSVAITWLMGSLTSQKVIYLVMIVLIVPAYSISCIGVSNRDLVEGMSGGTWVDVVLEHGGCVTEMAQDKPTVDIELVTMTVSNMAEVRSYCYEASLSDMASASRCPTQGEPSLDKQSDTQSVCKRTLGDRGWGNGCGIFGKGSLVTCSKFTCCKKMPGKSIQPENLEYRIMLPVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQSAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA ArD128000|AHL43502MKNPKRKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI 78LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKERKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMXXXXXGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHRLVRKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWLKKGSSIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGVHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA ArD158084|AHL43504MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI 79LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKERKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA H/PF/2013|AHZ13508MKNPKKKSGGFRIVNMLKRGVARVSPFGGLKRLPAGLLLGHGPIRMVLAI 80LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKEKKRRGADTSVGIVGLLLTTAMAAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA MR766_NIID|BAP47441MKNPKKKSGGFRIVNMLKRGVARVNPLGGLKRLPAGLLLGHGPIRMVLAI 81LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKERKRRGADTSIGIIGLLLTTAMAAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMTVNDIGYETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGKLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKIPVQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA prMEVEVTKKGDTYYMFADKKDAGKVVTFETESGPNRCSIQAMDIGHMCPATMS 82ABI54480_SouthAfrica YECPVLEPQYEPEDVDCWCNSTAAWIVYGTCTHKTTGETRRSRRSITLPSHASQKLETRSSTWLESREYSKYLIKVENWILRNPGYALVAAVIGWTLGSSRSQKIIFVTLLMLVAPAYSIRCIGIGNRDFIEGMSGGTWVDIVLEHGGCVTVMSNDKPTLDFELVTTTASNMAEVRSYCYEANISEMASDSRCPTQGEAYLDKMADSQFVCKRGYVDRGWGNGCGLFGKGSIVTCAKFTCVKKLTGKSIQPENLEYRVLVSVHASQHGGMINNDTNHQHDKENRARIDITASAPRVEVELGSFGSFSMECEPRSGLNFGDLYYLTMNNKHWLVNRDWFHDLSLPWHTGATSNNHHWNNKEALVEFREAHAKKQTAVVLGSQEGAVHAALAGALEAESDGHKATIYSGHLKCRLKLDKLRLKGMSYALCTGAFTFARTPSETIHGTATVELQYAGEDGPCKVPIVITSDTNSMASTGRLITANPVVTESGANSKMMVEIDPPFGDSYIIVGTGTTKITHHWHRAGSSIGRAFEATMRGAKRMAVLGDTAWDFGSVGGMFNSVGKFVHQVFGSAFKALFGGMSWFTQLLIGFLLIWMGLNARGGTVAMSFMGIGAMLIFLATSVSG prMEAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS 83AAV34151_Uganda_NHP YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIGYETDEDRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKIPVQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISL TCLALGGVMIFLSTAVSAprME AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS 84AHZ13508_FrenchPoly_2013YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA prMEAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS 85 gAHL43504YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA prMEAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS 86 AHL43503YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGETRRSRRSVSLRYHYTRKLQTRSQTWLESREYKKHLIMVENWIFRNPGFAIVSVAITWLMGSLTSQKVIYLVMIVLIVPAYSISCIGVSNRDLVEGMSGGTWVDVVLEHGGCVTEMAQDKPTVDIELVTMTVSNMAEVRSYCYEASLSDMASASRCPTQGEPSLDKQSDTQSVCKRTLGDRGWGNGCGIFGKGSLVTCSKFTCCKKMPGKSIQPENLEYRIMLPVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQSAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA prMEAAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATM 87 AHL43502SYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMXXXXXGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHRLVRKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWLKKGSSIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGVHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA prMEAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS 88 AHL43501YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCQHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAVCTAAKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA prMEAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS 89 AHL43500YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSSTSQKVIYLIMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDXXXXXXXNRAEVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA prMEAEITRRGSAYYMYLDRSDAGKAISFATNLGVNKCHVQIMDLGHMCDATMS 90 AHF49785YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGVHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA prMEAEITRRGSAYYMYLDRSDAGKAISFATNLGVNKCHVQIMDLGHMCDATMS 91 AHF49784_1976YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTC LALGGVMIFLSTAVSA prMEAEITRRGSAYYMYLDRSDAGKAISFATNLGVNKCHVQIMDLGHMCDATMS 92 AHF49783YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALAAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDIGHETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGVHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA prMEVEVTRRGSAYYMYLDRSDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS 93ACD75819_Micronesia YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA prMEAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS 94 ABI54475YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFTLVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTC LALGGVMIFLSTAVSA prMEAEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS 95 YP_002790881YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIGYETDEDRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKIPVQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISL TCLALGGVMIFLSTAVSAprME AEITRRGSAYYMYLDRSDAGKAISFATTLGVNKCHVQIMDLGHMCDATMS 96 BAP4744YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIKVENWIFRNPGFALVAVAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFTCSKKMTGKSIQPENLEYRIMLSVHGSQHSGMTVNDIGYETDENRAKVEVTPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGKLFSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKVPAETLHGTVTVEVQYAGTDGPCKIPVQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGDKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGVFNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVMIFLSTAVSA prMEAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS 97KU365780_2015_Brazil_isolate_BeH815744YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA prMEAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS 98KU365779_2015_Brazil_isolate_BeH819966YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA prMEAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS 99KU365778_2015_Brazil_isolate_BeH819015YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA prMEAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS 100KU365777_2015_Brazil_isolate_BeH818995YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA prMEAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS 101KU321639_2015_Brazil_isolate_ZikaSPH2015YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDIVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA prMEAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHTCDATMS 102KU312312_2015_Suriname_isolate_Z1106033YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNAKNGSISLMCLALGGVLIFLSTAVSA Premembrane/membraneAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS 103 proteinYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHKU321639_2015_Brazil_isolate_ZikaSPH2015STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST SQKVIYLVMILLIAPAYSEnvelop protein IRCIGVSNRDFVEGMSGGTWVDIVLEHGGCVTVMAQDKPTVDIELVTTTV 104KU321639_2015_Brazil_isolate_ZikaSPH2015SNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLST AVSA Capsid proteinMKNPKKKSGGFRIVNMLKRGVARVSPFGGLKRLPAGLLLGHGPIRMVLAI 105KU321639_2015_Brazil_isolate_ZikaSPH2015LAFLRFTAIKPSLGLINRWGSVGKKEAMEIIKKFKKDLAAMLRIINARKEKKRRGADTSVGIVGLLLTTAMAAEV Non-structuralVGCSVDFSKKETRCGTGVFVYNDVEAWRDRYKYHPDSPRRLAAAVKQAWE 106 protein 1DGICGISSVSRMENIMWRSVEGELNAILEENGVQLTVVVGSVKNPMWRGPKU321639_2015_Brazil_isolate_ZikaSPH2015QRLPVPVNELPHGWKAWGKSHFVRAAKTNNSFVVDGDTLKECPLKHRAWNSFLVEDHGFGVFHTSVWLKVREDYSLECDPAVIGTAVKGKEAVHSDLGYWIESEKNDTWRLKRAHLIEMKTCEWPKSHTLWTDGIEESDLIIPKSLAGPLSHHNTREGYRTQMKGPWHSEELEIRFEECPGTKVHVEETCGTRGPSLRSTTASGRVIEEWCCRECTMPPLSFRAKDGCWYGMEIRPRKEPESNLVRSMVT AGSTDHMDHFSLNon-structural GVLVILLMVQEGLKKRMTTKIIISTSMAVLVAMILGGFSMSDLAKLAILM 107protein 2A GATFAEMNTGGDVAHLALIAAFKVRPALLVSFIFRANWTPRESMLLALASKU321639_2015_Brazil_isolate_ZikaSPH2015CLLQTAISALEGDLMVLINGFALAWLAIRAMVVPRTDNITLAILAALTPLARGTLLVAWRAGLATCGGFMLLSLKGKGSVKKNLPFVMALGLTAVRLVDP INVVGLLLLTRSGKRSWPNon-structural PSEVLTAVGLICALAGGFAKADIEMAGPMAAVGLLIVSYVVSGKSVDMYI 108protein 2B ERAGDITWEKDAEVTGNSPRLDVALDESGDFSLVEDDGPPMREIILKVVLKU321639_2015_Brazil_isolate_ZikaSPH2015MTICGMNPIAIPFAAGAWYVYVKTGKRSGALWDVPAPKEVKKGE Non-structuralTTDGVYRVMTRRLLGSTQVGVGVMQEGVFHTMWHVTKGSALRSGEGRLDP 109 protein 3YWGDVKQDLVSYCGPWKLDAAWDGHSEVQLLAVPPGERARNIQTLPGIFKKU321639_2015_Brazil_isolate_ZikaSPH2015TKDGDIGAVALDYPAGTSGSPILDKCGRVIGLYGNGVVIKNGSYVSAITQGRREEETPVECFEPSMLKKKQLTVLDLHPGAGKTRRVLPEIVREAIKTRLRTVILAPTRVVAAEMEEALRGLPVRYMTTAVNVTHSGTEIVDLMCHATFTSRLLQPIRVPNYNLYIMDEAHFTDPSSIAARGYISTRVEMGEAAAIFMTATPPGTRDAFPDSNSPIMDTEVEVPERAWSSGFDWVTDYSGKTVWFVPSVRNGNEIAACLTKAGKRVIQLSRKTFETEFQKTKHQEWDFVVTTDISEMGANFKADRVIDSRRCLKPVILDGERVILAGPMPVTHASAAQRRGRIGRNPNKPGDEYLYGGGCAETDEDHAHWLEARMLLDNIYLQDGLIASLYRPEADKVAAIEGEFKLRTEQRKTFVELMKRGDLPVWLAYQVASAGITYTDRRWCFDGTTNNTIMEDSVPAEVWTRHGEKRVLKPRWMDARVCSDHAALKSFKEFAAGKR GAA Non-structuralFGVMEALGTLPGHMTERFQEAIDNLAVLMRAETGSRPYKAAAAQLPETLE 110 protein 4ATIMLLGLLGTVSLGIFFVLMRNKGIGKMGFGMVTLGASAWLMWLSEIEPAKU321639_2015_Brazil_isolate_ZikaSPH2015RIACVLIVVFLLLVVLIPEPEKQRSPQDNQMAIIIMVAVGLLGLITA Non-structuralNELGWLERTKSDLSHLMGRREEGATMGFSMDIDLRPASAWAIYAALTTFI 111 protein 4BTPAVQHAVTTSYNNYSLMAMATQAGVLFGMGKGMPFYAWDFGVPLLMIGCKU321639_2015_Brazil_isolate_ZikaSPH2015YSQLTPLTLIVAIILLVAHYMYLIPGLQAAAARAAQKRTAAGIMKNPVVDGIVVTDIDTMTIDPQVEKKMGQVLLMAVAVSSAILSRTAWGWGEAGALITAATSTLWEGSPNKYWNSSTATSLCNIFRGSYLAGASLIYTVTRNAGLVKRRGGGTGETLGEKWKARLNQMSALEFYSYKKSGITEVCREEARRALKDGVATGGHAVSRGSAKLRWLVERGYLQPYGKVIDLGCGRGGWSYYAATIRKVQEVKGYTKGGPGHEEPVLVQSYGWNIVRLKSGVDVFHMAAEPCDTLLCDIGESSSSPEVEEARTLRVLSMVGDWLEKRPGAFCIKVLCPYTSTMMETLERLQRRYGGGLVRVPLSRNSTHEMYWVSGAKSNTIKSVSTTSQLLLGRMDGPRR PV Non-structuralKYEEDVNLGSGTRAVVSCAEAPNMKIIGNRIERIRSEHAETWFFDENHPY 112 protein 5RTWAYHGSYEAPTQGSASSLINGVVRLLSKPWDVVTGVTGIAMTDTTPYGKU321639_2015_Brazil_isolate_ZikaSPH2015QQRVFKEKVDTRVPDPQEGTRQVMSMVSSWLWKELGKHKRPRVCTKEEFINKVRSNAALGAIFEEEKEWKTAVEAVNDPRFWALVDKEREHHLRGECQSCVYNMMGKREKKQGEFGKAKGSRAIWYMWLGARFLEFEALGFLNEDHWMGRENSGGGVEGLGLQRLGYVLEEMSRIPGGRMYADDTAGWDTRISRFDLENEALITNQMEKGHRALALAIIKYTYQNKVVKVLRPAEKGKTVMDIISRQDQRGSGQVVTYALNTFTNLVVQLIRNMEAEEVLEMQDLWLLRRSEKVTNWLQSNGWDRLKRMAVSGDDCVVKPIDDRFAHALRFLNDMGKVRKDTQEWKPSTGWDNWEEVPFCSHHFNKLHLKDGRSIVVPCRHQDELIGRARVSPGAGWSIRETACLAKSYAQMWQLLYFHRRDLRLMANAICSSVPVDWVPTGRTTWSIHGKGEWMTTEDMLVVWNRVWIEENDHMEDKTPVTKWTDIPYLGKREDLWCGSLIGHRPRTTWAENIKNTVNMVRRIIGDEEKYMDYLSTQVRYLGEEGSTPG VL SignalMETPAQLLFLLLLWLPDTTGAEVTRRGSAYYMYLDRNDAGEAISFPTTLG 113 peptide_prM-EMNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDIVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA Signal peptide_EMETPAQLLFLLLLWLPDTTGIRCIGVSNRDFVEGMSGGTWVDIVLEHGGC 114VTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA IgE HC signalMDWTWILFLVAAATRVHSVEVTRRGSAYYMYLDRSDAGEAISFPTTLGMN 115 peptide_prM-E #1KCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCH(Brazil_isolate_ZikaSPH2015)HKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA IgE HC signalMDWTWILFLVAAATRVHSVEVTRRGSAYYMYLDRSDAGEAISFPTTLGMN 116 peptide_prM-E #1KCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCH (ACD75819_Micronesia)HKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA IgE HC signalMDWTWILFLVAAATRVHSTRRGSAYYMYLDRSDAGEAISFPTTLGMNKCY 117 peptide_prM-E #2IQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKK(Brazil_isolate_ZikaSPH2015)GEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA HuIgG_(k )signalMETPAQLLFLLLLWLPDTTGVEVTRRGSAYYMYLDRSDAGEAISFPTTLG 118 peptide_prME #1MNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGT(Brazil_isolate_ZikaSPH2015)CHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA HuIgG_(k )signalMETPAQLLFLLLLWLPDTTGTRRGSAYYMYLDRSDAGEAISFPTTLGMNK 119 peptide_prME #2CYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHH(Brazil_isolate_ZikaSPH2015)KKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA HuIgG_(k )signalMETPAQLLFLLLLWLPDTTGIRCIGVSNRDFVEGMSGGTWVDVVLEHGGC 120 peptide_EVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEA(Brazil_isolate_ZikaSPH2015)YLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA IgE HC signalMDWTWILFLVAAATRVHSTRRGSAYYMYLDRSDAGEAISFPTTLGMNKCY 121 peptide_prM-E #2IQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKK (ACD75819_Micronesia)GEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA HuIgG_(k )signalMETPAQLLFLLLLWLPDTTGVEVTRRGSAYYMYLDRSDAGEAISFPTTLG 122 peptide_prME #1,MNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGT (ACD75819_Micronesia)CHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA HuIgG_(k )signalMETPAQLLFLLLLWLPDTTGTRRGSAYYMYLDRSDAGEAISFPTTLGMNK 123 peptide_prME #2,CYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHH (ACD75819_Micronesia)KKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA HuIgG_(k )signalMETPAQLLFLLLLWLPDTTGIRCIGVSNRDFVEGMSGGTWVDVVLEHGGC 124 peptide_E,VTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEA (ACD75819_Micronesia)YLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA HuIgG_(k )signal METPAQLLFLLLLWLPDTTG 125peptide IgE heavy chain MDWTWILFLVAAATRVHS 126 epsilon-1 signal peptideZika_RIO-U1_JEVsp AEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS 127Zika PRME Strain YECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHascension id: STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTANG09399 SQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA Japanese MLGSNSGQRVVFTILLLLVAPAYS 128encephalitis PRM signal sequence Zika_RIO-U1_JEVspMLGSNSGQRVVFTILLLLVAPAYSAEVTRRGSAYYMYLDRNDAGEAISFP 129 Zika PRME StrainTTLGMNKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWV ascension id:VYGTCHHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVE ANG09399 with JEVNWIFRNPGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSN PRM signalRDFVEGMSGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRS sequenceYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA Zika_RIO-EVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMSY 130 U1¬_VSVgSpECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSHS Zika PRME StrainTRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSSTS ascension id:QKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVTV ANG09399MAQDKPTVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGS ISLMCLALGGVLIFLSTAVSAVSV g protein MKCLLYLAFLFIGVNCA 131 signal sequence Zika_RIO-MKCLLYLAFLFigVNCAEVTRRGSAYYMYLDRNDAGEAISFPTTLGMNKC 132 U1¬_VSVgSpYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHK Zika PRME StrainKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRNPG ascension id:FALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMS ANG09399 with VSVGGTWVDVVLEHGGCVTVMAQDKPTVDIELVTTTVSNMAEVRSYCYEASIS g protein signalDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTC sequenceAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVNDTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLMWLGLNTKNGSISLMCLALGGVLIFLSTAVSA ZIKA_PRME_DSP_N154AVEVTRRGSAYYMYLDRSDAGEAISFPTTLGMNKCYIQIMDLGHMCDATMS 133 (glycosylationYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCHHKKGEARRSRRAVTLPSH mutant)STRKLQTRSQTWLESREYTKHLIRVENWIFRNPGFALAAAAIAWLLGSST Zika PRME StrainSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEGMSGGTWVDVVLEHGGCVT ascension id:VMAQDKPAVDIELVTTTVSNMAEVRSYCYEASISDMASDSRCPTQGEAYL ACD75819DKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLVTCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVADTGHETDENRAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA ZIKA_PRME_DSP_N154AMDWTWILFLVAAATRVHSVEVTRRGSAYYMYLDRSDAGEAISFPTTLGMN 134 (glycosylationKCYIQIMDLGHMCDATMSYECPMLDEGVEPDDVDCWCNTTSTWVVYGTCH mutant withHKKGEARRSRRAVTLPSHSTRKLQTRSQTWLESREYTKHLIRVENWIFRN signal peptide)PGFALAAAAIAWLLGSSTSQKVIYLVMILLIAPAYSIRCIGVSNRDFVEG Zika PRME StrainMSGGTWVDVVLEHGGCVTVMAQDKPAVDIELVTTTVSNMAEVRSYCYEAS ascension id:ISDMASDSRCPTQGEAYLDKQSDTQYVCKRTLVDRGWGNGCGLFGKGSLV ACD75819 with IgETCAKFACSKKMTGKSIQPENLEYRIMLSVHGSQHSGMIVADTGHETDENR signal peptideAKVEITPNSPRAEATLGGFGSLGLDCEPRTGLDFSDLYYLTMNNKHWLVHKEWFHDIPLPWHAGADTGTPHWNNKEALVEFKDAHAKRQTVVVLGSQEGAVHTALAGALEAEMDGAKGRLSSGHLKCRLKMDKLRLKGVSYSLCTAAFTFTKIPAETLHGTVTVEVQYAGTDGPCKVPAQMAVDMQTLTPVGRLITANPVITESTENSKMMLELDPPFGDSYIVIGVGEKKITHHWHRSGSTIGKAFEATVRGAKRMAVLGDTAWDFGSVGGALNSLGKGIHQIFGAAFKSLFGGMSWFSQILIGTLLVWLGLNTKNGSISLTCLALGGVLIFLSTAVSA

TABLE 33 ZIKV NCBI Accession Numbers (Amino Acid Sequences) Name GenBankAccession polyprotein [Zika virus] YP_002790881.1 polyprotein [Zikavirus] BAP47441.1 polyprotein [Zika virus] AEN75263.1 polyprotein [Zikavirus] AHL43504.1 polyprotein [Zika virus] AEN75266.1 polyprotein [Zikavirus] AHF49784.1 polyprotein [Zika virus] AHF49783.1 polyprotein [Zikavirus] AHF49785.1 polyprotein [Zika virus] ABI54475.1 polyprotein [Zikavirus] AHL43501.1 polyprotein [Zika virus] AHL43500.1 polyprotein [Zikavirus] AHL43502.1 polyprotein [Zika virus] AEN75265.1 polyprotein [Zikavirus] AHL43503.1 polyprotein [Zika virus] AEN75264.1 polyprotein [Zikavirus] AHZ13508.1 polyprotein [Zika virus] ACD75819.1 polyprotein [Zikavirus] AFD30972.1 polyprotein [Zika virus] AAK91609.1 envelope protein[Zika virus] AHL43462.1 envelope protein [Zika virus] AHL43464.1envelope protein [Zika virus] AHL43461.1 envelope protein [Zika virus]AHL43460.1 envelope protein [Zika virus] AHL43463.1 envelope protein[Zika virus] AHL43444.1 envelope protein [Zika virus] AHL43451.1envelope protein [Zika virus] AHL43437.1 envelope protein [Zika virus]AHL43455.1 envelope protein [Zika virus] AHL43448.1 envelope protein[Zika virus] AHL43439.1 envelope protein [Zika virus] AHL43468.1 Eprotein [Zika virus] AIC06934.1 envelope protein [Zika virus] AHL43450.1envelope protein [Zika virus] AHL43442.1 envelope protein [Zika virus]AHL43458.1 envelope glycoprotein [Zika virus] AHL16749.1 envelopeprotein [Zika virus] AHL43453.1 envelope protein [Zika virus] AHL43443.1envelope protein [Zika virus] AHL43438.1 envelope protein [Zika virus]AHL43441.1 envelope protein [Zika virus] AHL43457.1 envelope protein[Zika virus] AAK91609.1 polyprotein [Zika virus] AHL43505.1

Example 35 Surface Expressed DENV2 prME Antigens

The DENV2 prME polypeptide antigen sequences provided in Table 34 weretested to confirm that the DENV prME protein antigen is translated,properly folded and expressed on the surface of cells. For thepolypeptide sequences, the bolded sequence is Dengue signal sequence,the underlined sequence is DENV2 precursor membrane sequence, and theunmarked sequence is DENV2 envelope sequence. The sequences encoding thepolypeptides are codon-optimized. HeLa cells were transfected with DNAencoding the prMEs from nine different Dengue 2 isolates. After 24hours, surface expression of the prME was detected using three differentantibodies followed by goat-anti-human AF700 secondary antibody andsubjecting the cells to FACS analyses. Each of the three antibodies arebroadly neutralizing DENV2 prME antibodies that have in vivo efficacyagainst Dengue virus. D88 binds to DIII of Envelope protein for all 4Dengue serotypes (US20150225474). 2D22 binds to DIII of Envelope proteinfor Dengue 2 serotype. 5J7 binds to 3 domains of Envelope protein forDengue 3 serotype. FIG. 34B shows that two of the DENV2 prME antigensare recognized by the D88 and 2D22 antibodies. These results show thatthe two DENV2 prME antigens identified as Thailand/01 68/1979 andPeru/IQT29 13/1996 are expressed at the cell surface in aconformationally correct form and are excellent vaccine candidates (FIG.34A). FIG. 34B shows a repeat of staining in triplicate and in twodifferent cell lines (HeLa and 293T). These results confirm properconformation of expressed DENV2 prME antigens (in particular, the prMEantigens from Thailand/01 68/1979 and Peru/IQT29 13/1996) and alsoevidence at least non-inferior and even superior DENV2 antigenicity ascompared to Dengvaxia (CYD-TDV), a live attenuated tetravalent chimericvaccine. Antigen expressed from the mRNA encoding Dengue 2 prME fromPeru/IQT2913/1996 shows the best binding to 2 different DENV2 antibodiesin 293T cells and in HeLa cells (D88—binds all 4 serotypes 2D22—bindsDengue 2). This construct has a single amino acid difference from theDengue 2 Envelope III Domain immunodeterminant region (see bold,underline in SEQ ID NO: 168, Table 34).

TABLE 34 Example DENV2 PrME Polypeptide Sequence Polypeptide Name 5′ UTRORF 3′ UTR Sequence Dengue 2 TCAAGCTT ATGCTGAATATTCTGAACCGCCG TGATAATAMLNILNRRRRTA prME TTGGACCC CCGCCGGACTGCCGGGATTATAA GGCTGGAG GIIIMMIPTVMA(Thailand/ TCGTACAG TTATGATGATTCCCACCGTGATG CCTCGGTG FHLTTRNGEPHM0168/1979) AAGCTAAT GCCTTCCACCTGACCACCCGGAA GCCATGCT IVSRQEKGKSLLACGACTCA CGGGGAACCACATATGATCGTGT TCTTGCCC FKTEDGVNMCTL CTATAGGGCCAGACAGGAGAAGGGAAAGTCC CTTGGGCC MAMDLGELCEDT AAATAAGACTGCTGTTCAAGACCGAGGACGG TCCCCCCA ITYKCPLLRQNE GAGAAAAGCGTGAACATGTGCACCCTCATGG GCCCCTCC PEDIDCWCNSTS AAGAGTAACTATGGACCTGGGCGAACTCTGC TCCCCTTC TWVTYGTCTTTG GAAGAAATGAGGACACCATCACCTACAAGTG CTGCACCC EHRREKRSVALV ATAAGAGCCCCCCTGTTGAGGCAGAACGAGC GTACCCCC PHVGMGLETRTE CACCCGGAGGATATTGACTGCTGGTGC GTGGTCTT TWMSSEGAWKHA (SEQ IDAATTCGACCAGCACCTGGGTCAC TGAATAAA QRIETWILRHPG NO: 135)CTACGGGACTTGCACCACAACCG GTCTGAGT FTIMAAILAYTI GAGAACATCGGCGCGAAAAGCGCGGGCGGC GTTHFQRALIFI AGCGTGGCTTTGGTGCCTCACGT (SEQ ID LLTAVAPSMTMRCGGAATGGGGCTGGAGACTAGAA NO: 153) CIGISNRDFVEG CCGAGACTTGGATGTCGTCGGAAVSGGSWVDIVLE GGGGCCTGGAAACACGCACAGCG HGSCVTTMAKNKCATCGAAACTTGGATACTCAGGC PTLDFELIKTEA ATCCCGGCTTCACCATTATGGCCKQPATLRKYCIE GCGATCCTGGCATACACCATCGG AKLTNTTTESRCTACTACCCACTTCCAACGGGCCC PTQGEPSLNEEQ TGATCTTTATCCTCCTGACCGCTDKRFVCKHSMVD GTCGCACCATCCATGACCATGCG RGWGNGCGLFGKGTGTATCGGTATCAGCAACAGGG GGIVTCAMFTCK ACTTCGTGGAGGGAGTGTCGGGAKNMEGKIVQPEN GGATCCTGGGTGGATATTGTGCT LEYTIVVTPHSGGGAACACGGTTCCTGCGTCACTA EEHAVGNDTGKH CCATGGCGAAGAACAAGCCTACCGKEIKVTPQSSI CTGGACTTTGAGCTGATCAAAAC TEAELTGYGTVTTGAGGCCAAGCAGCCGGCCACCC MECSPRTGLDFN TGCGCAAGTACTGCATCGAAGCCEMVLLQMENKAW AAGCTGACCAATACCACTACCGA LVHRQWFLDLPLATCCCGCTGTCCGACCCAAGGGG PWLPGADTQGSN AGCCCTCCCTGAATGAGGAGCAGWIQKETLVTFKN GACAAGCGCTTCGTCTGCAAGCA PHAKKQDVVVLGTTCAATGGTCGACCGCGGCTGGG SQEGAMHTALTG GAAACGGCTGCGGACTGTTCGGAATEIQMSSGNLL AAGGGCGGCATTGTGACCTGTGC FTGHLKCRLRMDCATGTTCACTTGCAAGAAGAACA KLQLKGMSYSMC TGGAAGGAAAGATCGTGCAGCCCTGKFKVVKEIAE GAAAACCTGGAGTATACCATCGT TQHGTIVIRVQYCGTGACCCCGCACTCCGGGGAAG EGDGSPCKIPFE AACACGCTGTGGGAAACGACACCIMDLEKRHVLGR GGAAAGCACGGAAAGGAGATCAA LITVNPIVTEKDAGTGACCCCACAGTCGAGCATTA SPVNIEAEPPFG CCGAGGCCGAACTTACTGGTTACDSYIIIGVEPGQ GGCACTGTGACGATGGAATGTTC LKLNWFKKGSSIACCGAGAACTGGACTGGATTTCA GQMFETTMRGAK ACGAAATGGTGCTGCTCCAAATGRMAILGDTAWDF GAAAACAAGGCCTGGCTGGTGCA GSLGGVFTSIGKCCGCCAGTGGTTTCTTGACCTCC ALHQVFGAIYGA CTCTCCCTTGGCTGCCTGGAGCAAFSGVSWTMKIL GACACTCAGGGTTCCAACTGGAT IGVIITWIGMNSTCAGAAGGAAACACTCGTGACCT RSTSLSVSLVLV TCAAGAACCCTCACGCGAAGAAGGIVTLYLGVMVQ CAGGATGTGGTCGTGCTGGGAAG A (SEQ ID CCAGGAGGGAGCGATGCATACCGNO: 162) CCCTCACCGGCGCGACGGAGATT CAGATGTCCAGCGGAAACCTTCTGTTCACCGGACACCTGAAGTGCA GACTGAGGATGGACAAGCTGCAG CTCAAGGGAATGTCCTACTCCATGTGCACTGGAAAGTTCAAGGTCG TGAAGGAGATTGCCGAAACTCAG CATGGTACCATCGTGATCCGGGTGCAATATGAAGGGGACGGATCCC CGTGCAAGATCCCTTTCGAAATC ATGGACTTGGAGAAGCGACACGTGCTGGGCAGACTGATCACAGTCA ACCCCATCGTGACTGAGAAGGAT TCACCCGTGAACATTGAAGCCGAGCCGCCTTTCGGCGATAGCTACA TCATCATTGGCGTGGAACCGGGA CAGCTTAAGCTCAACTGGTTCAAGAAGGGTTCCTCGATCGGTCAAA TGTTTGAAACCACGATGCGGGGT GCCAAACGGATGGCCATTCTGGGAGACACCGCCTGGGATTTCGGCT CCTTGGGCGGAGTGTTCACTTCT ATCGGAAAGGCGCTGCACCAAGTGTTCGGAGCCATCTACGGCGCCG CGTTCTCGGGCGTCAGCTGGACC ATGAAGATTCTGATCGGGGTCATCATCACTTGGATTGGGATGAACT CACGGTCCACCTCCCTGAGCGTG TCCCTTGTCCTGGTCGGCATCGTGACCCTGTACCTCGGAGTGATGG TGCAGGCTTAG (SEQ ID NO: 144) Dengue 2 TCAAGCTTATGCTTAACATTCTCAACCGCCG TGATAATA MLNILNRRRRTA prME TTGGACCCCCGGAGAACTGCTGGTATTATCA GGCTGGAG GIIIMMIPTVMA (Thailand/ TCGTACAGTTATGATGATTCCCACTGTGATG CCTCGGTG FHLTTRNGEPHM 16681/1984) AAGCTAATGCCTTCCACCTGACCACGCGGAA GCCATGCT IVGRQEKGKSLL ACGACTCACGGCGAACCCCATATGATTGTCG TCTTGCCC FKTEDGVNMCTL CTATAGGGGTCGGCAGGAAAAGGGGAAGTCC CTTGGGCC MAIDLGELCEDT AAATAAGACTGCTGTTCAAAACTGAGGACGG TCCCCCCA ITYKCPLLRQNE GAGAAAAGAGTGAACATGTGCACCCTCATGG GCCCCTCC PEDIDCWCNSTS AAGAGTAACTATTGACCTGGGAGAGCTGTGC TCCCCTTC TWVTYGTCATTG GAAGAAATGAAGATACTATCACGTACAAGTG CTGCACCC EHRREKRSVALV ATAAGAGCCCCCCTGCTGCGCCAGAACGAGC GTACCCCC PHVGMGLETRTE CACCCTGAGGACATTGACTGCTGGTGC GTGGTCTT TWMSSEGAWKHV (SEQ IDAACTCCACGTCAACCTGGGTCAC TGAATAAA QRIETWILRHPG NO: 136)CTACGGAACTTGCGCGACTACCG GTCTGAGT FTIMAAILAYTI GCGAACATCGCAGAGAAAAGAGAGGGCGGC GTTHFQRALIFI AGCGTGGCCCTCGTGCCGCACGT (SEQ ID LLTAVAPSMTMRCGGGATGGGGCTGGAAACCCGGA NO: 154) CIGMSNRDFVEG CCGAAACCTGGATGTCCTCGGAAVSGGSWVDIVLE GGCGCCTGGAAGCACGTGCAGAG HGSCVTTMAKNKGATCGAAACTTGGATCCTCCGGC PTLDFELIKTEA ACCCGGGATTCACCATCATGGCCKQPATLRKYCIE GCCATCCTCGCTTACACAATCGG AKLTNTTTESRCAACCACTCACTTTCAACGCGCCC PTQGEPSLNEEQ TGATCTTCATCCTGCTTACCGCCDKRFVCKHSMVD GTGGCCCCGTCCATGACCATGCG RGWGNGCGLFGKCTGCATTGGAATGTCAAACCGGG GGIVTCAMFRCK ACTTCGTCGAGGGAGTCTCCGGAKNMEGKVVQPEN GGAAGCTGGGTGGACATCGTGCT LEYTIVITPHSGGGAGCACGGCAGCTGTGTGACCA EEHAVGNDTGKH CCATGGCCAAGAACAAGCCAACTGKEIKITPQSST CTTGATTTCGAACTGATCAAGAC TEAELTGYGTVTCGAGGCCAAGCAGCCTGCCACTC MECSPRTGLDFN TGAGGAAGTACTGTATCGAAGCGEMVLLQMENKAW AAGCTGACCAACACCACTACCGA LVHRQWFLDLPLATCCCGCTGCCCGACCCAGGGCG PWLPGADTQGSN AACCTTCCTTGAACGAAGAACAGWIQKETLVTFKN GACAAGAGATTCGTGTGCAAGCA PHAKKQDVVVLGTAGCATGGTCGACAGGGGATGGG SQEGAMHTALTG GGAACGGATGTGGACTCTTTGGGATEIQMSSGNLL AAGGGCGGAATCGTCACCTGTGC FTGHLKCRLRMDGATGTTCCGGTGCAAGAAGAACA KLQLKGMSYSMC TGGAGGGGAAGGTCGTGCAGCCCTGKFKVVKEIAE GAAAATCTCGAGTACACTATCGT TQHGTIVIRVQYGATCACCCCGCATTCCGGAGAGG EGDGSPCKIPFE AGCACGCCGTGGGCAACGACACCIMDLEKRHVLGR GGGAAGCACGGAAAGGAGATCAA LITVNPIVTEKDAATTACCCCTCAATCCTCCACCA SPVNIEAEPPFG CCGAAGCCGAATTGACTGGTTACDSYIIIGVEPGQ GGTACCGTGACTATGGAGTGCTC LKLNWFKKGSSIGCCGCGGACTGGCTTGGACTTCA GQMFETTMRGAK ACGAGATGGTGCTGCTGCAAATGRMAILGDTAWDF GAGAACAAGGCCTGGCTGGTGCA GSLGGVFTSIGKCCGGCAGTGGTTTCTTGATCTGC ALHQVFGAIYGA CTCTGCCTTGGCTGCCCGGAGCCAFSGVSWTMKIL GACACCCAGGGTAGCAATTGGAT IGVIITWIGMNSCCAGAAAGAGACACTCGTGACCT RSTSLSVTLVLV TTAAGAACCCGCACGCAAAGAAGGIVTLYLGVMVQ CAGGATGTCGTGGTCCTGGGAAG A (SEQ ID CCAAGAAGGGGCAATGCATACCGNO: 163) CACTCACTGGAGCCACTGAAATC CAGATGTCCTCCGGCAATCTGCTGTTCACCGGCCATCTGAAGTGCC GACTGCGCATGGACAAGCTCCAG CTTAAGGGAATGTCCTACTCCATGTGTACTGGAAAGTTCAAAGTCG TGAAGGAAATTGCCGAAACCCAG CACGGCACCATAGTGATCCGGGTGCAGTACGAGGGCGACGGCTCAC CCTGCAAAATCCCGTTCGAGATT ATGGATCTCGAAAAGCGCCACGTGCTGGGCAGACTGATTACCGTGA ACCCTATCGTGACCGAGAAGGAT TCCCCAGTGAACATCGAGGCCGAACCGCCCTTCGGAGACTCGTATA TCATCATCGGCGTGGAGCCCGGC CAGCTGAAGCTGAACTGGTTCAAGAAGGGGTCGAGCATCGGCCAGA TGTTCGAGACTACCATGCGCGGC GCGAAGAGGATGGCGATCCTGGGGGATACCGCTTGGGACTTCGGTT CCCTCGGCGGGGTGTTCACCTCG ATTGGGAAGGCCCTCCACCAAGTGTTCGGTGCAATCTACGGAGCGG CGTTCAGCGGAGTGTCGTGGACC ATGAAGATTCTGATCGGCGTGATCATCACCTGGATTGGCATGAACT CCCGGTCTACTAGCCTGTCGGTG ACCCTGGTGCTGGTCGGAATCGTGACCTTGTACCTGGGAGTGATGG TGCAAGCTTAG (SEQ ID NO: 145) Dengue 2 TCAAGCTTATGCTGAACATCCTGAACCGCAG TGATAATA MLNILNRRRRTA prME TTGGACCCAAGGAGAACCGCCGGTATTATTA GGCTGGAG GIIIMMIPTVMA (Jamaica/1409/ TCGTACAGTTATGATGATCCCCACCGTGATG CCTCGGTG FHLTTRNGEPHM 1983) AAGCTAATGCATTCCACCTGACTACCCGCAA GCCATGCT IVGRQEKGKSLL ACGACTCACGGAGAGCCGCATATGATCGTGG TCTTGCCC FKTEDGVNMCTL CTATAGGGGCCGCCAGGAAAAGGGAAAGTCC CTTGGGCC MAIDLGELCEDT AAATAAGACTGCTGTTCAAGACTGAGGACGG TCCCCCCA ITYKCPLLRQNE GAGAAAAGCGTGAACATGTGCACTCTCATGG GCCCCTCC PEDIDCWCNSTS AAGAGTAACCATCGACCTCGGCGAACTGTGC TCCCCTTC TWVTYGTCATTG GAAGAAATGAGGACACCATTACTTACAAGTG CTGCACCC EHRREKRSVALV ATAAGAGCCCCGCTGCTGAGACAGAACGAGC GTACCCCC PHVGMGLETRTE CACCCTGAGGACATCGACTGTTGGTGT GTGGTCTT TWMSSEGAWKHV (SEQ IDAACTCGACCTCCACCTGGGTCAC TGAATAAA QRIETWILRHPG NO: 137)CTACGGAACGTGCGCCACAACCG GTCTGAGT FTIMAAILAYTI GAGAACACCGCCGGGAAAAGCGGGGGCGGC GTTHFQRALIFI AGCGTGGCTCTGGTGCCGCACGT (SEQ ID LLTAVAPSMTMRCGGAATGGGTCTGGAGACTAGAA NO: 155) CIGISNRDFVEG CCGAAACCTGGATGTCATCCGAGVSGGSWVDIVLE GGGGCATGGAAACATGTGCAGCG HGSCVTTMAKNKAATCGAGACTTGGATCCTGAGAC PTLDFELIKTEA ACCCGGGCTTCACTATCATGGCGKQPATLRKYCIE GCCATCCTTGCCTACACCATTGG AKLTNTTTESRCCACTACTCACTTCCAACGGGCGC PTQGEPSLNEEQ TGATCTTCATACTGCTCACCGCGDKRFLCKHSMVD GTGGCCCCCTCCATGACGATGCG RGWGNGCGLFGKCTGCATCGGAATCTCCAACCGGG GGIVTCAMFTCK ACTTCGTGGAGGGCGTCAGCGGAKNMEGKVVLPEN GGCAGCTGGGTGGACATCGTGTT LEYTIVITPHSGGGAGCACGGAAGCTGCGTGACCA EEHAVGNDTGKH CCATGGCCAAGAACAAGCCCACTGKEIKITPQSSI CTTGATTTTGAGCTGATCAAGAC TEAELTGYGTVTGGAAGCAAAGCAGCCGGCCACTC MECSPRTGLDFN TGAGGAAGTACTGCATCGAGGCCEMVLLQMEDKAW AAGCTCACCAACACAACCACCGA LVHRQWFLDLPLATCTCGGTGCCCGACCCAAGGAG PWLPGADTQGSN AGCCATCACTGAACGAGGAACAGWIQKETLVTFKN GACAAGAGATTCCTGTGCAAACA PHAKKQDVVVLGTTCGATGGTGGACAGGGGATGGG SQEGAMHTALTG GAAATGGTTGCGGCCTGTTCGGCATEIQMSSGNLL AAAGGAGGCATTGTGACCTGTGC FTGHLKCRLRMDGATGTTCACTTGCAAGAAAAACA KLQLKGMSYSMC TGGAGGGGAAGGTCGTGTTGCCGTGKFKIVKEIAE GAGAACCTGGAGTACACTATCGT TQHGTIVIRVQYGATTACCCCGCACTCCGGGGAGG EGDGSPCKIPFE AACATGCCGTGGGAAATGACACCIMDLEKRHVLGR GGAAAGCACGGGAAGGAAATCAA LITVNPIVTEKDAATCACGCCTCAGTCCTCAATCA SPVNIEAEPPFG CCGAAGCCGAGCTTACCGGCTACDSYIIIGVEPGQ GGTACCGTGACCATGGAGTGCAG LKLNWFKKGSSICCCTCGGACTGGACTGGACTTCA GQMFETTMRGAK ACGAGATGGTGCTGCTGCAAATGRMAILGDTAWDF GAAGATAAGGCCTGGCTGGTGCA GSLGGVFTSIGKCCGGCAGTGGTTCTTGGATTTGC ALHQVFGAIYGA CACTGCCTTGGCTGCCCGGCGCGAFSGVSWTMKIL GATACCCAGGGTTCCAACTGGAT IGVIITWIGMNSTCAGAAGGAAACCCTCGTGACCT RSTSLSVSLVLV TCAAGAATCCTCACGCCAAGAAGGVVTLYLGAMVQ CAGGACGTGGTGGTGCTGGGTTC A (SEQ ID CCAAGAAGGGGCCATGCATACTGNO: 164) CCCTCACTGGAGCGACCGAAATC CAGATGTCGTCCGGCAACCTCCTGTTCACCGGCCACCTGAAGTGCC GCCTGCGGATGGACAAGTTGCAG CTGAAGGGAATGAGCTACTCGATGTGTACCGGAAAGTTCAAGATCG TGAAGGAAATCGCCGAAACCCAG CACGGAACCATCGTCATTAGAGTGCAGTACGAAGGGGACGGCAGCC CGTGCAAGATCCCCTTCGAAATT ATGGACCTGGAGAAGCGCCACGTGCTCGGAAGGCTCATCACTGTCA ACCCAATCGTCACCGAAAAGGAC TCCCCTGTGAACATCGAAGCAGAGCCCCCTTTCGGGGACTCCTACA TTATTATCGGCGTGGAGCCCGGC CAGCTGAAGCTGAACTGGTTCAAGAAGGGATCCTCGATCGGACAGA TGTTCGAAACCACCATGCGGGGA GCCAAGCGGATGGCTATTCTGGGAGATACCGCTTGGGATTTCGGCT CCCTCGGCGGCGTCTTTACTTCC ATCGGGAAAGCGCTCCACCAAGTGTTTGGAGCCATCTACGGTGCCG CTTTTTCCGGGGTGTCATGGACC ATGAAGATTCTTATCGGGGTCATTATTACTTGGATCGGCATGAACT CCCGGAGCACCTCGCTGTCCGTG AGCCTCGTGCTCGTGGGGGTGGTCACTCTGTATCTTGGTGCCATGG TGCAGGCCTAG (SEQ ID NO: 146) Dengue 2 TCAAGCTTATGCTTAACATCCTGAATAGAAG TGATAATA MLNILNRRRRTA prME TTGGACCCAAGAAGAACCGCCGGCATTATCA GGCTGGAG GIIIMMIPTVMA (Thailand/ TCGTACAGTTATGATGATACCCACCGTGATG CCTCGGTG FHLTTRNGEPHM NGS- AAGCTAATGCCTTCCACCTGACTACTCGCAA GCCATGCT IVSRQEKGKSLL C/1944) ACGACTCACGGAGAGCCTCATATGATCGTGT TCTTGCCC FKTEDGVNMCTL CTATAGGGCGCGGCAGGAGAAGGGAAAGTCC CTTGGGCC MAMDLGELCEDT AAATAAGACTGCTGTTTAAGACGGAGGACGG TCCCCCCA ITYKCPFLKQNE GAGAAAAGCGTGAACATGTGCACTCTTATGG GCCCCTCC PEDIDCWCNSTS AAGAGTAACAATGGACCTTGGAGAGCTGTGC TCCCCTTC TWVTYGTCTTTG GAAGAAATGAGGATACCATCACCTACAAGTG CTGCACCC EHRREKRSVALV ATAAGAGCTCCGTTCCTGAAGCAAAACGAGC GTACCCCC PHVGMGLETRTE CACCCTGAGGATATTGACTGCTGGTGC GTGGTCTT TWMSSEGAWKHA (SEQ IDAACTCCACCTCAACCTGGGTCAC TGAATAAA QRIETWILRHPG NO: 138)ATATGGGACCTGTACCACTACTG GTCTGAGT FTIMAAILAYTI GCGAACACCGCCGCGAGAAAAGAGGGCGGC GTTHFQRALIFI AGCGTGGCGTTGGTGCCTCACGT (SEQ ID LLTAVAPSMTMRCGGCATGGGTCTGGAAACTCGGA NO: 156) CIGISNRDFVEG CCGAAACTTGGATGAGCTCAGAGVSGGSWVDIVLE GGGGCATGGAAGCACGCCCAGAG HGSCVTTMAKNKGATTGAAACCTGGATTCTGCGCC PTLDFELIETEA ACCCTGGATTCACCATCATGGCGKQPATLRKYCIE GCTATTCTGGCGTACACTATTGG AKLTNTTTDSRCAACCACCCACTTTCAGCGGGCCC PTQGEPSLNEEQ TTATCTTCATCCTCCTCACTGCCDKRFVCKHSMVD GTGGCGCCCTCCATGACTATGCG RGWGNGCGLFGKGTGTATCGGAATTTCCAACCGCG GGIVTCAMFTCK ACTTCGTGGAAGGAGTGTCCGGAKNMKGKVVQPEN GGCTCCTGGGTCGACATTGTGCT LEYTIVITPHSGGGAACATGGTTCATGCGTGACCA EEHAVGNDTGKH CGATGGCCAAGAACAAGCCCACCGKEIKITPQSSI CTCGACTTCGAGCTGATCGAGAC TEAELTGYGTVTTGAAGCCAAGCAGCCGGCCACTC MECSPRTGLDFN TGCGGAAGTACTGTATCGAGGCCEMVLLQMENKAW AAGCTCACCAACACCACCACCGA LVHRQWFLDLPLTTCCCGCTGCCCGACCCAAGGAG PWLPGADTQGSN AACCTTCGCTCAACGAGGAGCAGWIQKETLVTFKN GACAAGCGGTTCGTGTGCAAGCA PHAKKQDVVVLGCAGCATGGTCGACAGGGGATGGG SQEGAMHTALTG GGAATGGATGCGGTCTGTTCGGAATEIQMSSGNLL AAGGGAGGCATTGTGACTTGTGC FTGHLKCRLRMDAATGTTCACTTGCAAGAAGAACA KLQLKGMSYSMC TGAAGGGGAAGGTCGTGCAGCCGTGKFKVVKEIAE GAAAACCTGGAGTACACCATCGT TQHGTIVIRVQYGATCACCCCTCATTCGGGCGAAG EGDGSPCKIPFE AACACGCTGTGGGGAATGATACCIMDLEKRHVLGR GGAAAGCACGGAAAGGAAATTAA LITVNPIVTEKDGATCACACCCCAATCCAGCATCA SPVNIEAEPPFG CTGAGGCAGAACTGACCGGCTACDSYIIIGVEPGQ GGCACTGTGACCATGGAGTGCTC LKLNWFKKGSSIGCCTCGGACTGGCCTGGACTTCA GQMIETTMRGAK ACGAGATGGTGCTGCTCCAAATGRMAILGDTAWDF GAAAACAAGGCCTGGCTGGTGCA GSLGGVFTSIGKCAGACAGTGGTTCCTCGATTTGC ALHQVFGAIYGA CCTTGCCGTGGCTCCCTGGCGCCAFSGVSWIMKIL GACACCCAGGGATCTAACTGGAT IGVIITWIGMNSCCAGAAGGAAACCCTTGTGACCT RSTSLSVSLVLV TCAAGAACCCGCACGCTAAGAAAGVVTLYLGVMVQ CAGGATGTGGTGGTGCTGGGAAG A (SEQ ID CCAGGAAGGAGCAATGCATACCGNO: 165) CGCTCACGGGTGCCACCGAGATC CAGATGAGCTCCGGGAACCTCCTGTTCACCGGTCACCTGAAGTGCC GACTCCGCATGGACAAACTGCAG CTCAAGGGGATGTCCTACTCCATGTGCACCGGGAAATTCAAGGTCG TGAAGGAGATCGCTGAGACTCAG CACGGTACTATCGTGATCCGGGTGCAGTATGAGGGAGATGGGAGCC CGTGCAAAATCCCATTTGAGATC ATGGACTTGGAAAAGCGCCATGTGCTGGGTCGGCTGATTACCGTGA ACCCAATCGTCACCGAAAAGGAC AGCCCCGTCAACATTGAAGCCGAACCACCCTTCGGAGACTCGTACA TCATCATTGGCGTGGAACCGGGC CAGCTGAAGCTGAACTGGTTCAAAAAGGGGTCCTCTATCGGCCAAA TGATCGAAACCACCATGCGGGGA GCTAAGCGGATGGCGATTTTGGGAGACACTGCGTGGGACTTTGGCT CACTGGGGGGAGTGTTCACCAGC ATCGGCAAAGCCCTGCACCAAGTGTTCGGTGCCATCTACGGAGCCG CCTTCAGCGGAGTGTCCTGGATC ATGAAGATCCTGATCGGCGTGATCATTACCTGGATCGGCATGAACT CCAGGTCCACCTCGCTCTCCGTG TCGCTGGTGCTGGTCGGGGTCGTGACCCTGTACCTGGGAGTGATGG TCCAGGCCTGA (SEQ ID NO: 147) Dengue 2 TCAAGCTTATGTTGAATATCCTGAACCGCCG TGATAATA MLNILNRRRRTA prME TTGGACCCCCGGAGAACTGCCGGAATTATCA GGCTGGAG GIIIMMIPTVMA (PuertoRico/ TCGTACAGTTATGATGATCCCTACCGTGATG CCTCGGTG FHLTTRNGEPHM PR159- AAGCTAATGCGTTCCACCTTACTACCCGGAA GCCATGCT IVSRQEKGKSLL S1/1969) ACGACTCACGGGGAGCCTCACATGATCGTGT TCTTGCCC FKTKDGTNMCTL CTATAGGGCACGCCAGGAGAAGGGGAAATCC CTTGGGCC MAMDLGELCEDT AAATAAGACTGCTGTTCAAGACCAAGGACGG TCCCCCCA ITYKCPFLKQNE GAGAAAAGTACCAACATGTGTACCCTGATGG GCCCCTCC PEDIDCWCNSTS AAGAGTAACGATGGACCTCGGAGAGCTGTGC TCCCCTTC TWVTYGTCTTTG GAAGAAATGAGGACACCATCACCTACAAATG CTGCACCC EHRREKRSVALV ATAAGAGCCCCGTTCCTGAAGCAGAACGAGC GTACCCCC PHVGMGLETRTE CACCCGGAAGATATTGACTGTTGGTGC GTGGTCTT TWMSSEGAWKHA (SEQ IDAACTCCACCTCCACTTGGGTCAC TGAATAAA QRIETWILRHPG NO: 139)CTACGGAACTTGCACCACTACTG GTCTGAGT FTIMAAILAYTI GGGAGCATAGACGGGAGAAGCGCGGGCGGC GTTHFQRVLIFI TCCGTGGCCCTGGTGCCGCACGT (SEQ ID LLTAIAPSMTMRCGGCATGGGACTGGAAACCAGAA NO: 157) CIGISNRDFVEG CCGAGACTTGGATGTCCAGCGAAVSGGSWVDIVLE GGCGCCTGGAAGCACGCCCAGCG HGSCVTTMAKNKGATTGAAACTTGGATCCTGAGGC PTLDFELIKTEA ACCCGGGTTTTACCATTATGGCCKQPATLRKYCIE GCTATCTTGGCATACACCATCGG AKLTNTTTDSRCCACCACCCACTTCCAACGCGTCC PTQGEPTLNEEQ TGATCTTCATCCTGCTGACCGCCDKRFVCKHSMVD ATTGCGCCCTCCATGACCATGCG RGWGNGCGLFGKGTGCATCGGAATCAGCAACCGCG GGIVTCAMFTCK ACTTCGTGGAAGGCGTCAGCGGCKNMEGKIVQPEN GGTTCTTGGGTGGACATCGTGTT LEYTVVITPHSGGGAGCACGGATCGTGCGTGACCA EEHAVGNDTGKH CCATGGCCAAGAACAAGCCGACCGKEVKITPQSSI CTCGATTTCGAGCTGATCAAGAC TEAELTGYGTVTTGAAGCCAAGCAGCCAGCTACCC MECSPRTGLDFN TGCGGAAGTATTGCATCGAAGCCEMVLLQMKDKAW AAGCTCACTAATACTACGACCGA LVHRQWFLDLPLCAGCCGGTGTCCGACCCAAGGAG PWLPGADTQGSN AGCCCACCCTGAATGAGGAACAAWIQKETLVTFKN GACAAGCGCTTCGTGTGCAAGCA PHAKKQDVVVLGTTCCATGGTGGACCGGGGCTGGG SQEGAMHTALTG GAAACGGCTGCGGACTGTTCGGGATEIQMSSGNLL AAAGGAGGAATTGTGACTTGCGC FTGHLKCRLRMDCATGTTCACTTGCAAGAAGAACA KLQLKGMSYSMC TGGAGGGGAAGATCGTCCAGCCTTGKFKVVKEIAE GAGAACCTCGAGTACACGGTCGT TQHGTIVIRVQYGATTACTCCGCACTCGGGAGAAG EGDGSPCKTPFE AACACGCCGTGGGCAACGACACCIMDLEKRHVLGR GGAAAGCATGGGAAGGAAGTGAA LTTVNPIVTEKDAATCACGCCCCAATCGTCGATTA SPVNIEAEPPFG CCGAGGCTGAGCTGACCGGCTACDSYIIIGVEPGQ GGCACCGTGACCATGGAGTGCTC LKLDWFKKGSSICCCGAGGACCGGACTGGACTTCA GQMFETTMRGAK ACGAAATGGTGCTGCTGCAGATGRMAILGDTAWDF AAGGACAAGGCCTGGCTGGTGCA GSLGGVFTSIGKCCGCCAGTGGTTCCTCGACCTCC ALHQVFGAIYGA CACTCCCCTGGCTGCCCGGAGCGAFSGVSWTMKIL GATACGCAGGGATCCAACTGGAT IGVIITWIGMNSCCAGAAGGAAACTCTTGTGACCT RSTSLSVSLVLV TCAAGAACCCTCATGCCAAGAAGGIVTLYLGVMVQ CAGGACGTGGTGGTCCTGGGATC A (SEQ ID CCAAGAGGGCGCGATGCACACCGNO: 166) CACTGACCGGCGCCACCGAAATT CAGATGTCCTCCGGAAACCTCCTGTTCACTGGCCACCTGAAGTGCA GACTCCGCATGGACAAGCTGCAG CTCAAGGGGATGAGCTACTCCATGTGTACCGGAAAATTCAAGGTCG TGAAGGAAATTGCAGAAACACAG CATGGGACAATTGTCATTCGGGTCCAGTACGAGGGCGATGGTTCAC CGTGCAAGACTCCATTCGAGATC ATGGATCTGGAGAAAAGACACGTGCTGGGTCGGCTGACTACCGTGA ACCCAATCGTGACTGAGAAGGAC TCCCCCGTGAACATCGAAGCCGAGCCTCCTTTTGGCGATTCCTACA TCATCATTGGAGTGGAACCCGGA CAGCTTAAGTTGGATTGGTTCAAGAAGGGCTCCTCGATCGGACAGA TGTTCGAAACCACCATGCGCGGT GCCAAGCGAATGGCCATCCTGGGGGACACCGCCTGGGACTTCGGTA GCCTGGGCGGAGTGTTTACCTCA ATTGGAAAGGCTCTGCACCAAGTGTTTGGGGCGATCTACGGAGCGG CCTTCAGCGGTGTCTCCTGGACT ATGAAGATTCTCATCGGAGTGATAATCACCTGGATCGGCATGAACA GCCGGTCAACCAGCCTGTCCGTG TCCCTGGTGCTGGTCGGCATCGTGACTCTCTACCTCGGAGTGATGG TGCAGGCCTAG (SEQ ID NO: 148) Dengue 2 TCAAGCTTATGCTCAACATACTGAACAGACG TGATAATA MLNILNRRRRTA prME TTGGACCCGAGAAGGACCGCCGGTATTATTA GGCTGGAG GIIIMMIPTVMA (16681- TCGTACAGTCATGATGATCCCTACTGTGATG CCTCGGTG FHLTTRNGEPHM PDK53) AAGCTAATGCATTCCACCTGACAACCCGCAA GCCATGCT IVSRQEKGKSLL ACGACTCACGGAGAGCCCCACATGATCGTGT TCTTGCCC FKTEVGVNMCTL CTATAGGGCACGCCAGGAGAAAGGGAAGTCA CTTGGGCC MAMDLGELCEDT AAATAAGACTGCTGTTCAAGACCGAAGTCGG TCCCCCCA ITYKCPLLRQNE GAGAAAAGCGTGAACATGTGTACCCTGATGG GCCCCTCC PEDIDCWCNSTS AAGAGTAACGATGGATCTTGGCGAACTGTGC TCCCCTTC TWVTYGTCTTMG GAAGAAATGAGGACACCATCACGTACAAGTG CTGCACCC EHRREKRSVALV ATAAGAGCCCCCCTGTTGCGGCAAAACGAAC GTACCCCC PHVGMGLETRTE CACCCAGAGGACATCGACTGCTGGTGT GTGGTCTT TWMSSEGAWKHV (SEQ IDAACTCCACCTCGACCTGGGTCAC TGAATAAA QRIETWILRHPG NO: 140)CTACGGAACCTGTACCACTATGG GTCTGAGT FTMMAAILAYTI GGGAACACCGGCGGGAGAAGCGCGGGCGGC GTTHFQRALILI TCCGTGGCGCTCGTGCCTCATGT (SEQ ID LLTAVTPSMTMRCGGCATGGGACTGGAGACTCGGA NO: 158) CIGMSNRDFVEG CTGAAACCTGGATGTCGTCGGAGVSGGSWVDIVLE GGGGCCTGGAAGCACGTCCAGCG HGSCVTTMAKNKGATCGAGACTTGGATCCTTCGCC PTLDFELIKTEA ATCCGGGCTTCACCATGATGGCCKQPATLRKYCIE GCCATCCTGGCCTACACCATCGG AKLTNTTTESRCAACCACCCATTTCCAACGGGCCC PTQGEPSLNEEQ TGATCCTGATCCTGTTGACTGCCDKRFVCKHSMVD GTGACCCCCTCCATGACTATGCG RGWGNGCGLFGKGTGCATTGGGATGTCGAACAGGG GGIVTCAMFRCK ATTTCGTGGAGGGAGTCAGCGGTKNMEGKVVQPEN GGCAGCTGGGTGGACATCGTGCT LEYTIVITPHSGGGAACATGGATCCTGCGTGACTA EEHAVGNDTGKH CCATGGCAAAGAACAAGCCAACCGKEIKITPQSSI CTCGATTTCGAACTGATCAAGAC TEAELTGYGTITCGAGGCGAAACAGCCGGCGACCC MECSPRTGLDFN TGAGGAAGTACTGCATCGAGGCCEIVLLQMENKAW AAGCTCACCAACACCACTACCGA LVHRQWFLDLPLGAGCAGATGCCCTACCCAAGGGG PWLPGADTQGSN AACCTTCCCTGAACGAGGAGCAGWIQKETLVTFKN GACAAGAGATTCGTCTGCAAGCA PHAKKQDVVVLGCTCCATGGTGGACCGCGGCTGGG SQEGAMHTALTG GAAACGGATGCGGACTCTTCGGAATEIQMSSGNLL AAGGGCGGTATTGTGACCTGTGC FTGHLKCRLRMDCATGTTCCGCTGCAAGAAAAACA KLQLKGMSYSMC TGGAAGGGAAAGTGGTGCAGCCCTGKFKVVKEIAE GAGAACCTCGAGTACACTATCGT TQHGTIVIRVQYGATCACACCGCACAGCGGAGAAG EGDGSPCKIPFE AACACGCCGTGGGCAACGACACTIMDLEKRHVLGR GGAAAGCACGGGAAGGAAATCAA LITVNPIVTEKDGATCACCCCGCAATCCTCAATCA SPVNIEAEPPFG CTGAGGCTGAGTTGACCGGCTACDSYIIIGVEPGQ GGGACTATTACCATGGAATGCTC LKLNWFKKGSSICCCACGAACGGGACTGGACTTCA GQMFETTMRGAK ACGAAATTGTGTTGCTCCAAATGRMAILGDTAWDF GAAAACAAGGCCTGGCTCGTGCA GSLGGVFTSIGKCCGGCAGTGGTTCCTGGATCTGC ALHQVFGAIYGA CCCTGCCGTGGCTGCCGGGTGCCAFSGVSWTMKIL GACACTCAGGGGAGCAACTGGAT IGVIITWIGMNSTCAGAAGGAAACCCTTGTGACCT RSTSLSVTLVLV TCAAGAACCCCCACGCAAAGAAGGIVTLYLGVMVQ CAGGACGTGGTGGTGCTGGGTAG A (SEQ ID CCAAGAAGGCGCCATGCACACGGNO: 167) CCCTGACCGGAGCGACCGAGATC CAGATGTCCAGCGGAAATCTGCTCTTTACTGGTCATCTGAAGTGCA GACTTCGGATGGACAAGCTGCAA CTGAAGGGAATGTCCTACTCAATGTGCACTGGAAAGTTCAAGGTCG TGAAGGAGATCGCCGAAACCCAG CACGGGACTATCGTCATCCGCGTGCAGTACGAAGGAGATGGCTCCC CGTGCAAGATCCCTTTCGAAATC ATGGACCTGGAGAAGCGCCACGTGTTGGGGCGCCTTATTACTGTGA ACCCCATCGTGACCGAGAAGGAC TCCCCTGTCAACATCGAGGCTGAACCGCCATTCGGAGATTCCTATA TCATTATCGGAGTGGAACCGGGC CAGCTCAAGCTGAATTGGTTCAAGAAGGGATCCTCGATTGGCCAGA TGTTCGAAACGACTATGCGGGGC GCTAAGCGCATGGCCATCCTGGGCGATACTGCCTGGGATTTTGGTT CTCTGGGCGGAGTGTTCACCTCC ATTGGAAAGGCCCTGCACCAAGTGTTCGGCGCCATCTACGGTGCCG CGTTTAGCGGTGTCTCATGGACC ATGAAAATCCTCATTGGCGTGATCATTACCTGGATTGGCATGAACT CCAGAAGCACTTCCCTGTCCGTG ACCCTGGTGCTCGTCGGAATTGTGACACTCTACCTCGGAGTGATGG TGCAGGCTTGA (SEQ ID NO: 149) Dengue 2 TCAAGCTTATGCTGAACATTTTGAACAGACG TCAAGCTT MLNILNRRRRTA prME TTGGACCCCCGAAGGACCGCAGGCATTATCA TTGGACCC GIIIMMIPTVMA (Peru/IQT2913/ TCGTACAGTTATGATGATCCCTACCGTGATG TCGTACAG FHLTTRNGEPHM 1996) AAGCTAATGCCTTCCATCTGACTACTAGGAA AAGCTAAT IVSRQEKGKSLL ACGACTCACGGAGAGCCACATATGATCGTGT ACGACTCA FKTKDGTNMCTL CTATAGGGCGCGCCAGGAAAAGGGAAAGAGC CTATAGGG MAMDLGELCEDT AAATAAGACTGCTTTTTAAAACCAAGGACGG AAATAAGA ITYKCPFLKQNE GAGAAAAGCACGAACATGTGCACCCTTATGG GAGAAAAG PEDIDCWCNSTS AAGAGTAACCATGGACCTGGGGGAGTTGTGC AAGAGTAA TWVTYGTCTTTG GAAGAAATGAGGACACCATCACCTACAAGTG GAAGAAAT EHRREKRSVALV ATAAGAGCCCCGTTCCTGAAGCAAAACGAGC ATAAGAGC PHVGMGLETRTE CACCCCGAAGATATTGACTGCTGGTGC CACC TWMSSEGAWKHA (SEQ IDAACTCCACCTCCACCTGGGTCAC (SEQ ID QRIETWILRHPG NO: 141)TTATGGGACTTGCACCACCACCG NO: 159) FTIMAAILAYTI GCGAACATCGCAGAGAAAAGAGAGTTHFQRVLIFI AGCGTGGCCCTGGTCCCCCACGT LLTAIAPSMTMRCGGGATGGGCCTCGAGACTCGGA CIGISNRDFVEG CCGAAACTTGGATGTCATCAGAGVSGGSWVDIVLE GGCGCATGGAAGCATGCTCAGCG HGSCVTTMAKNKGATCGAAACCTGGATCCTGAGAC PTLDFELIKTEA ACCCTGGTTTCACAATTATGGCCKQPATLRKYCIE GCCATTCTTGCGTACACGATCGG AKLTNTTTDSRCAACGACTCATTTCCAACGCGTGC PTQGEPTLNEEQ TGATCTTCATTCTCCTGACCGCTDKRFVCKHSMVD ATTGCGCCGTCCATGACTATGCG RGWGNGCGLFGKGTGCATCGGAATCTCAAACCGGG GGIVTCAMFTCK ACTTCGTGGAAGGAGTGTCGGGAKNMEGKIVQPEN GGATCCTGGGTGGACATTGTGCT LEYTVVITPHSGGGAGCACGGTTCCTGCGTCACCA EEHAVGNDTGKH CCATGGCCAAAAACAAGCCTACCGKEVKITPQSSI CTGGACTTCGAGCTGATCAAGAC TEAELTGYGTVTTGAGGCCAAGCAGCCCGCGACCC MECSPRTGLDFN TCCGGAAGTACTGCATCGAGGCCEMVLLQMEDKAW AAGTTGACCAACACTACTACCGA LVHRQWFLDLPLTTCCCGGTGCCCGACCCAAGGAG PWLPGADTQGSN AACCAACTCTGAACGAAGAACAGWIQKETLVTFKN GATAAGCGGTTTGTGTGCAAGCA PHAKKQDVVVLGCTCAATGGTGGACAGGGGATGGG SQEGAMHTALTG GCAACGGCTGTGGACTGTTCGGAATEIQMSSGNLL AAGGGTGGTATTGTGACCTGTGC FTGHLKCRLRMDAATGTTTACCTGTAAAAAGAATA KLQLKGMSYSMC TGGAGGGGAAGATCGTGCAGCCTTGKFKIVKEIAE GAAAATCTCGAGTACACTGTCGT TQHGTIVIRVQYCATCACCCCGCACTCGGGAGAGG EGDGSPCKIPFE AGCACGCTGTGGGCAACGACACCIMDLEKRHVLGR GGAAAGCACGGAAAGGAGGTCAA LITVNPIVTEKDGATAACCCCGCAATCCTCCATTA SPVNIEAEPPFG CGGAAGCCGAACTGACTGGTTAC DSYIIIG AEPGQ GGCACCGTGACTATGGAGTGCTC LKLDWFKKGSSI CCCTCGGACCGGCCTGGACTTCAGQMFETTMRGAK ACGAAATGGTGCTGCTCCAAATG RMAILGDTAWDFGAAGATAAGGCCTGGCTGGTGCA GSLGGVFTSIGK CAGGCAGTGGTTCCTGGATCTCCALHQVFGAIYGA CGCTGCCGTGGCTGCCTGGCGCT AFSGVSWTMKILGACACTCAGGGAAGCAACTGGAT IGVIITWIGMNS CCAGAAGGAAACCCTCGTGACCTRSTSLSVSLVLV TTAAGAACCCCCACGCCAAGAAG GIVTLYLGVMVQCAGGATGTGGTGGTGTTGGGAAG A (SEQ ID CCAGGAGGGGGCCATGCATACTG NO: 168)CCCTCACCGGCGCGACCGAAATC CAGATGTCGTCCGGCAATCTGCT GTTCACCGGACACCTCAAGTGTCGCCTTCGGATGGACAAGCTGCAG CTGAAGGGAATGAGCTACAGCAT GTGCACCGGGAAGTTCAAGATCGTGAAGGAAATCGCCGAAACCCAG CACGGAACCATCGTGATCCGGGT GCAGTACGAGGGCGACGGTTCTCCCTGCAAAATCCCCTTCGAAATC ATGGATCTGGAGAAGAGACACGT CCTGGGTCGCCTGATCACCGTGAACCCCATTGTGACTGAGAAGGAC TCCCCAGTGAACATCGAAGCGGA GCCCCCATTCGGAGACAGCTACATTATCATTGGTGCCGAACCGGGG CAGCTGAAACTGGACTGGTTCAA GAAGGGCAGCTCGATTGGCCAAATGTTCGAAACGACAATGCGGGGC GCAAAGCGCATGGCCATCCTGGG AGACACTGCCTGGGACTTCGGGTCCCTTGGGGGGGTGTTCACCTCG ATCGGAAAAGCCTTGCACCAAGT GTTCGGCGCAATCTACGGCGCCGCGTTCTCGGGAGTCTCCTGGACT ATGAAGATCCTGATCGGTGTCAT CATCACCTGGATCGGGATGAACTCCCGGTCCACTTCCCTCTCGGTG TCACTCGTGCTTGTGGGAATTGT CACCCTGTACCTCGGAGTGATGGTGCAGGCCTGA (SEQ ID NO: 150) Dengue 2 TCAAGCTT ATGCTGAATATTCTGAACCGACGTGATAATA MLNILNRRRRTA prME TTGGACCC CCGCCGCACTGCCGGAATCATTA GGCTGGAGGIIIMMIPTVMA (Thailand/ TCGTACAG TCATGATGATCCCTACCGTGATG CCTCGGTGFHLTTRNGEPHM PUO- AAGCTAAT GCGTTCCATCTCACCACTCGGAA GCCATGCT IVSRQEKGKSLL218/1980) ACGACTCA TGGCGAACCCCATATGATCGTGT TCTTGCCC FKTEDGVNMCTLCTATAGGG CGAGACAGGAAAAGGGAAAGAGC CTTGGGCC MAMDLGELCEDT AAATAAGACTTTTGTTCAAAACTGAAGATGG TCCCCCCA ITYKCPLLRQNE GAGAAAAGAGTGAACATGTGCACTCTCATGG GCCCCTCC PEDIDCWCNSTS AAGAGTAACAATGGATCTGGGCGAACTGTGC TCCCCTTC TWVTYGTCTTTG GAAGAAATGAAGATACCATCACTTACAAGTG CTGCACCC EHRREKRSVALV ATAAGAGCTCCGCTGTTGAGACAGAACGAGC GTACCCCC PHVGMGLETRTE CACCCTGAGGACATCGACTGCTGGTGT GTGGTCTT TWMSSEGAWKHA (SEQ IDAACAGCACTTCCACCTGGGTCAC TGAATAAA QRIEIWILRHPG NO: 142)CTACGGCACTTGCACTACCACCG GTCTGAGT FTIMAAILAYTI GAGAACACCGGCGCGAGAAGAGGGGGCGGC GTTHFQRALIFI AGCGTGGCTCTTGTGCCGCACGT (SEQ ID LLTAVAPSMTMRCGGCATGGGACTCGAGACTCGGA NO: 160) CIGISNRDFVEG CCGAAACCTGGATGTCATCCGAAVSGGSWVDIVLE GGAGCCTGGAAACACGCCCAACG HGSCVTTMAKNKGATCGAAATTTGGATCCTGAGAC PTLDFELIKTEA ACCCCGGTTTCACTATCATGGCCKQPATLRKYCIE GCAATCCTGGCGTACACTATTGG AKLTNTTTESRCCACCACGCACTTCCAGAGGGCCC PTQGEPSLNEEQ TCATTTTCATCCTCCTGACTGCCDKRFVCKHSMVD GTGGCGCCATCCATGACCATGAG RGWGNGCGLFGKATGTATTGGCATTTCCAACCGCG GGIVTCAMFTCK ATTTCGTGGAGGGAGTGTCCGGAKNMEGKVVQPEN GGATCCTGGGTCGACATCGTGCT LEYTIVVTPHSGGGAACACGGATCTTGCGTCACCA EEHAVGNDTGKH CCATGGCTAAGAACAAGCCCACCGKEIKVTPQSSI CTCGACTTCGAGCTGATCAAGAC TEAELTGYGTVTAGAAGCCAAGCAGCCGGCCACCC MECSPRTGLDFN TCCGCAAGTATTGCATTGAAGCCEMVLLQMENKAW AAGCTTACCAACACCACCACCGA LVHRQWFLDLPLGTCGCGGTGCCCAACCCAAGGAG PWLPGADTQGSN AGCCGAGCCTCAATGAGGAACAGWIQKETLVTFKN GACAAGCGCTTCGTGTGCAAACA PHAKKQDVVVLGCAGCATGGTCGACCGGGGTTGGG SQEGAMHTALTG GCAACGGATGTGGCCTGTTCGGGATEIQMSSGNLL AAGGGTGGCATTGTGACTTGCGC FTGHLKCRLRMDAATGTTCACTTGCAAGAAGAACA KLQLKGMSYSMC TGGAGGGGAAAGTGGTGCAACCCTGKFKVVKEIAE GAGAACCTGGAGTACACCATCGT TQHGTIVIRVQYCGTGACCCCACACTCCGGAGAGG EGDGSPCKIPFE AGCACGCCGTGGGAAACGACACGIMDLEKRHVLGR GGGAAGCATGGAAAGGAGATCAA LITVNPIVTEKDGGTCACACCCCAATCATCTATTA SPVNIEAEPPFG CCGAGGCCGAACTGACCGGATACDSYIIIGVEPGQ GGTACTGTGACGATGGAGTGCAG LKLNWFKKGSSICCCGAGGACTGGACTGGACTTCA GQMFETTMRGAK ACGAAATGGTGCTGCTGCAAATGRMAILGDTAWDF GAGAACAAGGCCTGGCTCGTGCA GSLGGVFTSIGKCCGGCAGTGGTTTCTGGATCTCC ALHQVFGAIYGA CACTGCCGTGGTTGCCGGGAGCCAFSGVSWTMKIL GACACCCAGGGGTCGAACTGGAT IGVIITWIGMNSCCAGAAGGAAACTCTTGTGACGT RSTSLSVSLVLV TTAAGAATCCTCACGCGAAGAAGGIVTLYLGVMVQ CAGGACGTGGTGGTCCTGGGATC A (SEQ ID GCAGGAAGGAGCTATGCACACCGNO: 169) CTCTGACCGGCGCCACTGAGATC CAGATGTCCTCGGGCAACCTCCTGTTCACCGGTCATCTGAAGTGCC GGCTGCGGATGGACAAATTGCAG CTGAAGGGGATGTCCTACTCCATGTGCACCGGGAAGTTCAAGGTCG TGAAGGAGATCGCGGAAACTCAG CACGGCACCATTGTCATTAGAGTGCAGTACGAGGGAGATGGTTCAC CGTGCAAGATACCGTTCGAAATC ATGGACCTGGAAAAGAGACATGTCTTGGGACGCCTGATCACTGTGA ACCCTATCGTGACCGAAAAGGAC TCCCCTGTGAACATCGAGGCGGAGCCGCCTTTCGGCGACTCCTACA TCATTATCGGAGTGGAGCCCGGG CAGCTGAAGCTCAACTGGTTTAAGAAGGGGTCCAGCATCGGCCAGA TGTTCGAAACCACCATGCGGGGG GCGAAGAGGATGGCGATCCTGGGAGACACCGCCTGGGATTTCGGTT CACTGGGCGGAGTGTTCACCTCC ATCGGAAAGGCCCTGCACCAAGTGTTCGGCGCAATCTACGGTGCTG CCTTCTCGGGAGTCTCCTGGACC ATGAAGATCCTGATCGGCGTGATTATCACATGGATCGGCATGAACA GCCGGTCAACCTCCCTTTCCGTG TCCCTGGTGCTGGTCGGCATCGTGACTCTGTACCTGGGCGTGATGG TGCAGGCCTGA (SEQ ID NO: 151) Dengue 2 TCAAGCTTATGCTGAACATTCTGAACCGGAG TGATAATA MLNILNRRRRTA prME TTGGACCCAAGAAGAACCGCCGGCATTATTA GGCTGGAG GIIIMMIPTVMA (D2Y98P) TCGTACAGTCATGATGATTCCCACTGTGATG CCTCGGTG FHLTTRNGEPHM with AAGCTAATGCATTTCACCTGACCACCCGGAA GCCATGCT IVSRQEKGKSLL native ACGACTCACGGAGAACCTCATATGATCGTGT TCTTGCCC FKTENGVNMCTL leader CTATAGGGCGAGACAGGAGAAGGGAAAGTCC CTTGGGCC MAMDLGELCEDT AAATAAGACTGCTGTTCAAGACAGAAAACGG TCCCCCCA ITYNCPLLRQNE GAGAAAAGAGTGAACATGTGCACCCTGATGG GCCCCTCC PEDIDCWCNSTS AAGAGTAACCATGGATCTCGGCGAACTGTGC TCCCCTTC TWVTYGTCTATG GAAGAAATGAGGATACTATCACCTACAACTG CTGCACCC EHRREKRSVALV ATAAGAGCTCCGTTGCTGCGCCAAAACGAGC GTACCCCC PHVGMGLETRTE CACCCGGAGGACATCGACTGCTGGTGT GTGGTCTT TWMSSEGAWKHA (SEQ IDAACTCCACGTCGACCTGGGTCAC TGAATAAA QRIETWVLRHPG NO: 143)CTACGGCACTTGCACCGCGACCG GTCTGAGT FTIMAAILAYTI GCGAACACAGAAGAGAGAAACGCGGGCGGC GTTYFQRVLIFI TCCGTCGCTCTGGTGCCGCACGT (SEQ ID LLTAVAPSMTMRCGGGATGGGGCTTGAAACCCGGA NO: 161) CIGISNRDFVEG CTGAAACCTGGATGAGCTCGGAGVSGGSWVDIVLE GGCGCTTGGAAGCATGCCCAGCG HGSCVTTMAKNKCATCGAAACTTGGGTGCTGAGGC PTLDFELIKTEA ATCCAGGCTTCACAATCATGGCCKHPATLRKYCIE GCCATCCTCGCGTACACCATCGG AKLTNTTTASRCTACTACGTACTTCCAGCGGGTGT PTQGEPSLNEEQ TGATCTTCATTCTGCTGACCGCCDKRFVCKHSMVD GTGGCCCCTAGCATGACCATGCG RGWGNGCGLFGKGTGCATCGGGATCTCCAACCGCG GGIVTCAMFTCK ATTTCGTGGAGGGGGTGTCCGGTKNMEGKIVQPEN GGAAGCTGGGTGGACATTGTGCT LEYTIVITPHSGGGAGCACGGCTCGTGCGTGACCA EENAVGNDTGKH CCATGGCCAAGAACAAGCCCACCGKEIKVTPQSSI CTTGATTTTGAGCTGATCAAGAC TEAELTGYGTVTCGAAGCGAAACACCCCGCGACCC MECSPRTGLDFN TCCGGAAGTACTGCATTGAAGCCEMVLLQMENKAW AAGCTCACCAACACTACCACGGC LVHRQWFLDLPLCTCCCGGTGCCCTACCCAAGGAG PWLPGADTQGSN AACCTTCCTTGAACGAAGAACAGWIQKETLVTFKN GACAAGCGCTTCGTGTGCAAGCA PHAKKQDVVVLGTTCAATGGTGGACCGGGGCTGGG SQEGAMHTALTG GAAATGGCTGTGGCCTCTTCGGAATEIQMSSGNLL AAAGGCGGAATTGTGACTTGCGC FTGHLKCRLRMDAATGTTCACTTGCAAGAAGAACA KLQLKGMSYSMC TGGAGGGAAAGATTGTGCAGCCCTGKFKVVKEIAE GAGAACCTCGAGTACACTATTGT TQHGTIVIRVQYCATCACTCCCCACTCCGGCGAAG EGDGSPCKIPFE AAAACGCTGTCGGCAACGACACCIMDLEKRHVLGR GGAAAGCATGGAAAGGAGATCAA LITVNPIVTEKDGGTCACCCCGCAATCCTCAATTA SPVNIEAEPPFG CTGAGGCAGAACTGACCGGTTACDSYIIIGVEPGQ GGAACTGTGACTATGGAGTGTTC LKLSWFKKGSSICCCTCGCACCGGCCTCGATTTCA GQMFETTMRGAK ACGAGATGGTGCTGCTGCAAATGRMAILGDTAWDF GAGAACAAGGCCTGGCTGGTGCA GSLGGVFTSIGKCCGGCAGTGGTTCCTCGATTTGC ALHQVFGAIYGA CCCTGCCGTGGCTGCCGGGAGCCAFSGVSWTMKIL GACACTCAGGGATCCAACTGGAT IGVVITWIGMNSCCAGAAAGAAACCCTCGTGACCT RSTSLSVSLVLV TCAAAAACCCCCACGCGAAGAAGGVVTLYLGVMVQ CAGGACGTGGTGGTGCTGGGTTC A (SEQ ID CCAAGAAGGGGCGATGCATACCGNO: 170) CCCTGACTGGTGCTACCGAAATC CAGATGTCAAGCGGAAATCTCCTGTTTACCGGTCACCTGAAGTGCA GGCTCCGGATGGACAAGTTGCAG CTGAAGGGGATGTCGTACAGCATGTGTACTGGGAAGTTCAAGGTCG TGAAGGAGATTGCCGAAACCCAG CACGGAACCATAGTCATCAGGGTCCAGTACGAGGGCGACGGCAGCC CTTGCAAGATCCCGTTCGAGATC ATGGATCTGGAGAAGCGACACGTGCTGGGCCGGCTTATCACTGTGA ATCCAATCGTGACCGAGAAAGAC TCGCCCGTGAACATCGAAGCCGAGCCGCCGTTCGGCGACTCATACA TCATCATCGGCGTGGAACCAGGA CAGCTGAAGCTGTCATGGTTCAAGAAGGGTTCCAGCATTGGTCAGA TGTTCGAAACAACGATGCGCGGA GCCAAGCGCATGGCTATCCTTGGGGACACCGCCTGGGACTTCGGGT CGCTGGGAGGAGTGTTTACCAGC ATCGGAAAGGCCCTGCACCAAGTGTTCGGTGCCATCTACGGAGCCG CATTTTCCGGAGTGTCGTGGACT ATGAAGATTCTGATCGGCGTCGTGATTACCTGGATCGGGATGAACT CCAGGTCTACTTCCCTCTCCGTG AGCCTGGTGCTGGTCGGCGTGGTCACCCTGTATCTGGGCGTGATGG TCCAGGCTTAG (SEQ ID NO: 152)

TABLE 35 Full-length Dengue Amino Acid Sequences (Homo sapiens strains;Brazil, Cuba and U.S.) GenBank Collection Accession Length Type CountryGenome Region Date Virus Name AGN94866 3392 1 Brazil UTR5CMENS1NS 2010Dengue virus 1 2ANS2BNS3NS4 isolate 12898/BR- A2KNS4BNS5UT PE/10,complete R3 genome AGN94867 3392 1 Brazil UTR5CMENS1NS 2010 Dengue virus1 2ANS2BNS3NS4 isolate 13501/BR- A2KNS4BNS5UT PE/10, complete R3 genomeAGN94868 3392 1 Brazil UTR5CMENS1NS 2010 Dengue virus 1 2ANS2BNS3NS4isolate 13671/BR- A2KNS4BNS5UT PE/10, complete R3 genome AGN94869 3392 1Brazil UTR5CMENS1NS 2010 Dengue virus 1 2ANS2BNS3NS4 isolate 13861/BR-A2KNS4BNS5UT PE/10, complete R3 genome AGN94870 3392 1 BrazilUTR5CMENS1NS 2010 Dengue virus 1 2ANS2BNS3NS4 isolate 14985/BR-A2KNS4BNS5UT PE/10, complete R3 genome AGN94871 3392 1 BrazilUTR5CMENS1NS 1996 Dengue virus 1 2ANS2BNS3NS4 isolate 21814/BR-A2KNS4BNS5UT PE/96, complete R3 genome AGN94872 3392 1 BrazilUTR5CMENS1NS 1997 Dengue virus 1 2ANS2BNS3NS4 isolate 40604/BR-A2KNS4BNS5UT PE/97, complete R3 genome AGN94873 3392 1 BrazilUTR5CMENS1NS 1997 Dengue virus 1 2ANS2BNS3NS4 isolate 41111/BR-A2KNS4BNS5UT PE/97, complete R3 genome AGN94874 3392 1 BrazilUTR5CMENS1NS 1998 Dengue virus 1 2ANS2BNS3NS4 isolate 52082/BR-A2KNS4BNS5UT PE/98, complete R3 genome AGN94875 3392 1 BrazilUTR5CMENS1NS 1999 Dengue virus 1 2ANS2BNS3NS4 isolate 59049/BR-A2KNS4BNS5UT PE/99, complete R3 genome AGN94876 3392 1 BrazilUTR5CMENS1NS 2000 Dengue virus 1 2ANS2BNS3NS4 isolate 70523/BR-A2KNS4BNS5UT PE/00, complete R3 genome AGN94877 3392 1 BrazilUTR5CMENS1NS 2001 Dengue virus 1 2ANS2BNS3NS4 isolate 74488/BR-A2KNS4BNS5UT PE/01, complete R3 genome AGN94878 3392 1 BrazilUTR5CMENS1NS 2001 Dengue virus 1 2ANS2BNS3NS4 isolate 75861/BR-A2KNS4BNS5UT PE/01, complete R3 genome AGN94879 3392 1 BrazilUTR5CMENS1NS 2002 Dengue virus 1 2ANS2BNS3NS4 isolate 88463/BR-A2KNS4BNS5UT PE/02, complete R3 genome AGN94865 3392 1 BrazilUTR5CMENS1NS 2010 Dengue virus 1 2ANS2BNS3NS4 isolate 9808/BR-A2KNS4BNS5UT PE/10, complete R3 genome ACO06150 3392 1 BrazilUTR5CMENS1NS 2000 Dengue virus 1 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT1/BR/BID- R3 V2374/2000, complete genome ACO06151 3392 1 BrazilUTR5CMENS1NS 2000 Dengue virus 1 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT1/BR/BID- R3 V2375/2000, complete genome ACO06153 3392 1 BrazilUTR5CMENS1NS 2001 Dengue virus 1 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT1/BR/BID- R3 V2378/2001, complete genome ACO06155 3392 1 BrazilUTR5CMENS1NS 2002 Dengue virus 1 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT1/BR/BID- R3 V2381/2002, complete genome ACO06157 3392 1 BrazilUTR5CMENS1NS 2003 Dengue virus 1 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT1/BR/BID- R3 V2384/2003, complete genome ACO06161 3392 1 BrazilUTR5CMENS1NS 2004 Dengue virus 1 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT1/BR/BID- R3 V2389/2004, complete genome ACO06164 3392 1 BrazilUTR5CMENS1NS 2005 Dengue virus 1 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT1/BR/BID- R3 V2392/2005, complete genome ACO06167 3392 1 BrazilUTR5CMENS1NS 2006 Dengue virus 1 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT1/BR/BID- R3 V2395/2006, complete genome ACO06170 3392 1 BrazilUTR5CMENS1NS 2007 Dengue virus 1 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT1/BR/BID- R3 V2398/2007, complete genome ACO06173 3392 1 BrazilUTR5CMENS1NS 2008 Dengue virus 1 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT1/BR/BID- R3 V2401/2008, complete genome ACY70762 3392 1 BrazilUTR5CMENS1NS 2008 Dengue virus 1 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS51/BR/BID- V3490/2008, complete genome ACJ12617 3392 1 BrazilUTR5CMENS1NS Dengue virus 1 2ANS2BNS3NS4 isolate DF01- A2KNS4BNS5UTHUB01021093, R3 complete genome AHC08447 3392 1 Brazil CMENS1NS2ANS 2011Dengue virus 1 2BNS3NS4A2KN strain S4BNS5 1266/2011/BR/RJ/ 2011polyprotein gene, partial cds AHC08446 3392 1 Brazil CMENS1NS2ANS 2010Dengue virus 1 2BNS3NS4A2KN strain S4BNS5 242/2010/BR/RJ/ 2010polyprotein gene, partial cds AHC08448 3392 1 Brazil CMENS1NS2ANS 1988Dengue virus 1 2BNS3NS4A2KN strain S4BNS5 36034/BR/RJ/ 1988 polyproteingene, partial cds AHC08449 3392 1 Brazil CMENS1NS2ANS 1989 Dengue virus1 2BNS3NS4A2KN strain S4BNS5 38159/BR/RJ/ 1989 polyprotein gene, partialcds AHC08450 3392 1 Brazil CMENS1NS2ANS 2000 Dengue virus 1 2BNS3NS4A2KNstrain S4BNS5 66694/BR/ES/ 2000 polyprotein gene, partial cds AHC084513392 1 Brazil CMENS1NS2ANS 2001 Dengue virus 1 2BNS3NS4A2KN strainS4BNS5 68826/BR/RJ/ 2001 polyprotein gene, partial cds AGN94880 3391 2Brazil UTR5CMENS1NS 2010 Dengue virus 2 2ANS2BNS3NS4 isolate 13858/BR-A2KNS4BNS5UT PE/10, complete R3 genome AGN94881 3391 2 BrazilUTR5CMENS1NS 2010 Dengue virus 2 2ANS2BNS3NS4 isolate 14905/BR-A2KNS4BNS5UT PE/10, complete R3 genome AGN94882 3391 2 BrazilUTR5CMENS1NS 2010 Dengue virus 2 2ANS2BNS3NS4 isolate 19190/BR-A2KNS4BNS5UT PE/10, complete R3 genome AGN94884 3391 2 BrazilUTR5CMENS1NS 1995 Dengue virus 2 2ANS2BNS3NS4 isolate 3275/BR-A2KNS4BNS5UT PE/95, complete R3 genome AGN94885 3391 2 BrazilUTR5CMENS1NS 1995 Dengue virus 2 2ANS2BNS3NS4 isolate 3311/BR-A2KNS4BNS5UT PE/95, complete R3 genome AGN94886 3391 2 BrazilUTR5CMENS1NS 1995 Dengue virus 2 2ANS2BNS3NS4 isolate 3337/BR-A2KNS4BNS5UT PE/95, complete R3 genome AGN94887 3391 2 BrazilUTR5CMENS1NS 1997 Dengue virus 2 2ANS2BNS3NS4 isolate 37473/BR-A2KNS4BNS5UT PE/97, complete R3 genome AGN94888 3391 2 BrazilUTR5CMENS1NS 1998 Dengue virus 2 2ANS2BNS3NS4 isolate 47913/BR-A2KNS4BNS5UT PE/98, complete R3 genome AGN94889 3391 2 BrazilUTR5CMENS1NS 1998 Dengue virus 2 2ANS2BNS3NS4 isolate 51347/BR-A2KNS4BNS5UT PE/98, complete R3 genome AGN94890 3391 2 BrazilUTR5CMENS1NS 1999 Dengue virus 2 2ANS2BNS3NS4 isolate 57135/BR-A2KNS4BNS5UT PE/99, complete R3 genome AGN94891 3391 2 BrazilUTR5CMENS1NS 2000 Dengue virus 2 2ANS2BNS3NS4 isolate 72144/BR-A2KNS4BNS5UT PE/00, complete R3 genome AGN94892 3391 2 BrazilUTR5CMENS1NS 2002 Dengue virus 2 2ANS2BNS3NS4 isolate 87086/BR-A2KNS4BNS5UT PE/02, complete R3 genome AGN94883 3391 2 BrazilUTR5CMENS1NS 2010 Dengue virus 2 2ANS2BNS3NS4 isolate 9479/BR-A2KNS4BNS5UT PE/10, complete R3 genome AGK36299 3391 2 BrazilCMENS1NS2ANS Mar. 30, 2010 Dengue virus 2 2BNS3NS4A2KN isolate ACS380,S4BNS5UTR3 complete genome AGK36289 3391 2 Brazil UTR5CMENS1NS Mar. 1,2010 Dengue virus 2 2ANS2BNS3NS4 isolate ACS46, A2KNS4BNS5UT completegenome R3 AGK36290 3391 2 Brazil UTR5CMENS1NS Mar. 1, 2010 Dengue virus2 2ANS2BNS3NS4 isolate ACS46_II, A2KNS4BNS5UT complete genome R3AGK36291 3391 2 Brazil UTR5CMENS1NS Apr. 12, 2010 Dengue virus 22ANS2BNS3NS4 isolate ACS538, A2KNS4BNS5UT complete genome R3 AGK362923391 2 Brazil UTR5CMENS1NS May 4, 2010 Dengue virus 2 2ANS2BNS3NS4isolate ACS542, A2KNS4BNS5UT complete genome R3 AGK36294 3391 2 BrazilUTR5CMENS1NS May 4, 2010 Dengue virus 2 2ANS2BNS3NS4 isolate ACS721,A2KNS4BNS5UT complete genome R3 ACO06152 3391 2 Brazil UTR5CMENS1NS 2000Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V2376/2000, complete genome AET43250 3391 2 Brazil CMENS1NS2ANS 2000Dengue virus 2 2BNS3NS4A2KN isolate DENV- S4BNS5UTR3 2/BR/BID-V2377/2000, complete genome ACO06154 3391 2 Brazil UTR5CMENS1NS 2001Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V2379/2001, complete genome ACO06156 3391 2 Brazil UTR5CMENS1NS 2002Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V2382/2002, complete genome ACW82928 3391 2 Brazil UTR5CMENS1NS 2003Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V2385/2003, complete genome ACO06158 3391 2 Brazil UTR5CMENS1NS 2003Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V2386/2003, complete genome ACO06162 3391 2 Brazil UTR5CMENS1NS 2004Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V2390/2004, complete genome ACO06165 3391 2 Brazil UTR5CMENS1NS 2005Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V2393/2005, complete genome ACO06168 3391 2 Brazil UTR5CMENS1NS 2006Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V2396/2006, complete genome ACO06171 3391 2 Brazil UTR5CMENS1NS 2007Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V2399/2007, complete genome ACS32031 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V2402/2008, complete genome ACW82873 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V3481/2008, complete genome ACW82874 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V3483/2008, complete genome ACW82875 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V3486/2008, complete genome ACY70763 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5 2/BR/BID-V3495/2008, complete genome ADI80655 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5 2/BR/BID-V3637/2008, complete genome ACY70778 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V3638/2008, complete genome ACY70779 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5 2/BR/BID-V3640/2008, complete genome ACY70780 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5 2/BR/BID-V3644/2008, complete genome ACY70781 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V3645/2008, complete genome ACY70782 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V3648/2008, complete genome ACY70783 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V3650/2008, complete genome ACY70784 3391 2 Brazil UTR5CMENS1NS 2008Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/BR/BID- R3V3653/2008, complete genome AGK36297 3391 2 Brazil UTR5CMENS1NS Apr. 15,2010 Dengue virus 2 2ANS2BNS3NS4 isolate DGV106, A2KNS4BNS5UT completegenome R3 AGK36295 3391 2 Brazil UTR5CMENS1NS Feb. 24, 2010 Dengue virus2 2ANS2BNS3NS4 isolate DGV34, A2KNS4BNS5UT complete genome R3 AGK362933391 2 Brazil UTR5CMENS1NS Feb. 24, 2010 Dengue virus 2 2ANS2BNS3NS4isolate DGV37, A2KNS4BNS5UT complete genome R3 AGK36298 3391 2 BrazilUTR5CMENS1NS Mar. 9, 2010 Dengue virus 2 2ANS2BNS3NS4 isolate DGV69,A2KNS4BNS5UT complete genome R3 AGK36296 3391 2 Brazil UTR5CMENS1NS Mar.24, 2010 Dengue virus 2 2ANS2BNS3NS4 isolate DGV91, A2KNS4BNS5UTcomplete genome R3 AFV95788 3391 2 Brazil CMENS1NS2ANS 2008 Dengue virus2 2BNS3NS4A2KN strain S4BNS5 BR0337/2008/RJ/ 2008 polyprotein gene,partial cds AFV95787 3391 2 Brazil CMENS1NS2ANS 2008 Dengue virus 22BNS3NS4A2KN strain S4BNS5 BR0450/2008/RJ/ 2008 polyprotein gene,partial cds ADV39968 3391 2 Brazil CMENS1NS2ANS 2008 Dengue virus 22BNS3NS4A2KN strain S4BNS5 BR0690/RJ/2008 polyprotein gene, complete cdsADV71220 3391 2 Brazil CMENS1NS2ANS 1990 Dengue virus 2 2BNS3NS4A2KNstrain S4BNS5 BR39145/RJ/90 polyprotein gene, partial cds ADV71215 33912 Brazil CMENS1NS2ANS 1990 Dengue virus 2 2BNS3NS4A2KN strain S4BNS5BR41768/RJ/90 polyprotein gene, partial cds ADV71216 3391 2 BrazilCMENS1NS2ANS 1991 Dengue virus 2 2BNS3NS4A2KN strain S4BNS5BR42727/RJ/91 polyprotein gene, partial cds ADV71217 3391 2 BrazilCMENS1NS2ANS 1994 Dengue virus 2 2BNS3NS4A2KN strain S4BNS5BR48622/CE/94 polyprotein gene, partial cds ADV71218 3391 2 BrazilCMENS1NS2ANS 1998 Dengue virus 2 2BNS3NS4A2KN strain S4BNS5BR61310/RJ/98 polyprotein gene, partial cds ADV71219 3391 2 BrazilCMENS1NS2ANS 1999 Dengue virus 2 2BNS3NS4A2KN strain S4BNS5BR64905/RJ/99 polyprotein gene, partial cds AFH53774 3390 2 BrazilUTR5CMENS1NS Dengue virus 2 2ANS2BNS3NS4 strain JHA1, A2KNS4BNS5 partialgenome AGN94893 3390 3 Brazil UTR5CMENS1NS 2003 Dengue virus 32ANS2BNS3NS4 isolate A2KNS4BNS5UT 101905/BR- R3 PE/03, complete genomeAGN94902 3390 3 Brazil UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate 129/BR- A2KNS4BNS5UT PE/04, complete R3 genome AGN94899 3390 3Brazil UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4 isolate 145/BR-A2KNS4BNS5UT PE/04, complete R3 genome AGN94903 3390 3 BrazilUTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4 isolate 161/BR-A2KNS4BNS5UT PE/04, complete R3 genome AGN94896 3390 3 BrazilUTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4 isolate 206/BR-A2KNS4BNS5UT PE/05, complete R3 genome AGN94904 3390 3 BrazilUTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4 isolate 249/BR-A2KNS4BNS5UT PE/05, complete R3 genome AGN94901 3390 3 BrazilUTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4 isolate 255/BR-A2KNS4BNS5UT PE/05, complete R3 genome AGN94905 3390 3 BrazilUTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4 isolate 263/BR-A2KNS4BNS5UT PE/05, complete R3 genome AGN94898 3390 3 BrazilUTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4 isolate 277/BR-A2KNS4BNS5UT PE/05, complete R3 genome AGN94906 3390 3 BrazilUTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4 isolate 283/BR-A2KNS4BNS5UT PE/05, complete R3 genome AGN94907 3390 3 BrazilUTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4 isolate 314/BR-A2KNS4BNS5UT PE/06, complete R3 genome AGN94897 3390 3 BrazilUTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4 isolate 339/BR-A2KNS4BNS5UT PE/05, complete R3 genome AGN94908 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate 411/BR-A2KNS4BNS5UT PE/06, complete R3 genome AGN94909 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate 418/BR-A2KNS4BNS5UT PE/06, complete R3 genome AGN94910 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate 420/BR-A2KNS4BNS5UT PE/06, complete R3 genome AGN94911 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate 423/BR-A2KNS4BNS5UT PE/06, complete R3 genome AGN94912 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate 424/BR-A2KNS4BNS5UT PE/06, complete R3 genome AGN94900 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate 603/BR-A2KNS4BNS5UT PE/06, complete R3 genome AGN94895 3390 3 BrazilUTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4 isolate 81257/BR-A2KNS4BNS5UT PE/02, complete R3 genome AGN94894 3390 3 BrazilUTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4 isolate 85469/BR-A2KNS4BNS5UT PE/02, complete R3 genome AFK83756 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate A2KNS4BNS5UTD3BR/ACN/2007, R3 complete genome AFK83755 3390 3 Brazil UTR5CMENS1NS2009 Dengue virus 3 2ANS2BNS3NS4 isolate A2KNS4BNS5UT D3BR/AL95/2009, R3complete genome AFK83754 3390 3 Brazil UTR5CMENS1NS 2004 Dengue virus 32ANS2BNS3NS4 isolate A2KNS4BNS5UT D3BR/BR8/04, R3 complete genomeAFK83753 3390 3 Brazil UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4isolate A2KNS4BNS5UT D3BR/BV4/02, R3 complete genome AFK83762 3390 3Brazil UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4 isolateA2KNS4BNS5UT D3BR/CU6/02, R3 complete genome AFK83759 3390 3 BrazilUTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4 isolate A2KNS4BNS5UTD3BR/MR9/03, R3 complete genome AFK83761 3390 3 Brazil UTR5CMENS1NS 2003Dengue virus 3 2ANS2BNS3NS4 isolate A2KNS4BNS5UT D3BR/PV1/03, R3complete genome AFK83760 3390 3 Brazil UTR5CMENS1NS 2002 Dengue virus 32ANS2BNS3NS4 isolate A2KNS4BNS5UT D3BR/SL3/02, R3 complete genomeAHG23238 3390 3 Brazil UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5 3/BR/BID- V2383/2002, complete genome ACO061593390 3 Brazil UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4 isolateDENV- A2KNS4BNS5UT 3/BR/BID- R3 V2387/2003, complete genome ACO061603390 3 Brazil UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4 isolateDENV- A2KNS4BNS5UT 3/BR/BID- R3 V2388/2003, complete genome ACO061633390 3 Brazil UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4 isolateDENV- A2KNS4BNS5UT 3/BR/BID- R3 V2391/2004, complete genome ACO061663390 3 Brazil UTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4 isolateDENV- A2KNS4BNS5UT 3/BR/BID- R3 V2394/2005, complete genome ACO061693390 3 Brazil UTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolateDENV- A2KNS4BNS5UT 3/BR/BID- R3 V2397/2006, complete genome ACO061723390 3 Brazil UTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolateDENV- A2KNS4BNS5UT 3/BR/BID- R3 V2400/2007, complete genome ACO061743390 3 Brazil UTR5CMENS1NS 2008 Dengue virus 3 2ANS2BNS3NS4 isolateDENV- A2KNS4BNS5UT 3/BR/BID- R3 V2403/2008, complete genome ACQ444853390 3 Brazil UTR5CMENS1NS 2001 Dengue virus 3 2ANS2BNS3NS4 isolateDENV- A2KNS4BNS5UT 3/BR/BID- R3 V2977/2001, complete genome ACQ444863390 3 Brazil UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4 isolateDENV- A2KNS4BNS5UT 3/BR/BID- R3 V2983/2003, complete genome ACY707433390 3 Brazil UTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolateDENV- A2KNS4BNS5UT 3/BR/BID- R3 V3417/2006, complete genome ACY707443390 3 Brazil UTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolateDENV- A2KNS4BNS5 3/BR/BID- V3423/2006, complete genome ACY70745 3390 3Brazil UTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV-A2KNS4BNS5UT 3/BR/BID- R3 V3424/2006, complete genome ACY70746 3390 3Brazil UTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV-A2KNS4BNS5 3/BR/BID- V3427/2006, complete genome ACY70747 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3429/2006, complete genome ACY70748 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3430/2006, complete genome ACW82870 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT3/BR/BID- R3 V3431/2006, complete genome ACY70749 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3434/2006, complete genome ACY70750 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3435/2006, complete genome ACY70751 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3441/2006, complete genome ACY70752 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3442/2006, complete genome ACW82871 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT3/BR/BID- R3 V3444/2006, complete genome ACY70753 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3446/2006, complete genome ACY70754 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT3/BR/BID- R3 V3451/2006, complete genome ACY70755 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT3/BR/BID- R3 V3456/2006, complete genome ACY70756 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT3/BR/BID- R3 V3457/2006, complete genome ACY70757 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3460/2006, complete genome ACW82872 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT3/BR/BID- R3 V3463/2006, complete genome ACY70758 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3464/2006, complete genome ACY70759 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3465/2006, complete genome ACY70760 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3469/2007, complete genome ACY70761 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3470/2007, complete genome ACY70764 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3584/2006, complete genome ACY70765 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3585/2007, complete genome ACY70766 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3588/2007, complete genome ACY70767 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3589/2007, complete genome ACY70768 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3590/2007, complete genome ACY70769 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3591/2007, complete genome ACY70770 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3593/2007, complete genome ACY70771 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT3/BR/BID- R3 V3597/2007, complete genome ACY70772 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3598/2007, complete genome ACY70773 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3601/2007, complete genome ACY70774 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3605/2007, complete genome ACY70775 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS53/BR/BID- V3606/2007, complete genome ACY70776 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT3/BR/BID- R3 V3609/2007, complete genome ACY70777 3390 3 BrazilUTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT3/BR/BID- R3 V3615/2007, complete genome AEV42062 3390 3 BrazilUTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4 isolate A2KNS4BNS5UTDENV3/BR/ R3 D3LIMHO/2006, complete genome AGH08164 3390 3 BrazilUTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4 strain 95016/BR-A2KNS4BNS5UT PE/02, complete R3 genome AEX91754 3387 4 BrazilUTR5CMENS1NS Sep. 8, 2010 Dengue virus 4 2ANS2BNS3NS4 isolateA2KNS4BNS5UT Br246RR/10, R3 complete genome AIQ84223 3387 4 BrazilUTR5CMENS1NS Mar. 28, 2012 Dengue virus 4 2ANS2BNS3NS4 strain DENV-4/MT/A2KNS4BNS5UT BR12_TVP17898/ R3 2012 isolate serum_12, complete genomeAIQ84224 3387 4 Brazil UTR5CMENS1NS Mar. 30, 2012 Dengue virus 42ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UT BR20_TVP17906/ R3 2012isolate serum_20, complete genome AIQ84225 3387 4 Brazil UTR5CMENS1NSMar. 30, 2012 Dengue virus 4 2ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UTBR23_TVP17909/ R3 2012 isolate serum_23, complete genome AIQ84226 3387 4Brazil UTR5CMENS1NS Apr. 19, 2012 Dengue virus 4 2ANS2BNS3NS4 strainDENV-4/MT/ A2KNS4BNS5UT BR24_TVP17910/ R3 2012 isolate serum_24,complete genome AIQ84227 3387 4 Brazil UTR5CMENS1NS Apr. 12, 2012 Denguevirus 4 2ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UT BR27_TVP17913/ R32012 isolate serum_27, complete genome AIQ84228 3387 4 BrazilUTR5CMENS1NS Apr. 19, 2012 Dengue virus 4 2ANS2BNS3NS4 strain DENV-4/MT/A2KNS4BNS5UT BR28_TVP17914/ R3 2012 isolate serum_28, complete genomeAIQ84220 3387 4 Brazil UTR5CMENS1NS Apr. 23, 2012 Dengue virus 42ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UT BR2_TVP17888/ R3 2012isolate serum_2, complete genome AIQ84245 3387 4 Brazil UTR5CMENS1NSApr. 20, 2012 Dengue virus 4 2ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UTBR33_TVP17919/ R3 2012 isolate serum_33, complete genome AIQ84244 3387 4Brazil UTR5CMENS1NS Mar. 30, 2012 Dengue virus 4 2ANS2BNS3NS4 strainDENV-4/MT/ A2KNS4BNS5UT BR35_TVP17921/ R3 2012 isolate serum_35,complete genome AIQ84243 3387 4 Brazil UTR5CMENS1NS Apr. 3, 2012 Denguevirus 4 2ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UT BR40_TVP17926/ R32012 isolate serum_40, complete genome AIQ84242 3387 4 BrazilUTR5CMENS1NS Apr. 5, 2012 Dengue virus 4 2ANS2BNS3NS4 strain DENV-4/MT/A2KNS4BNS5UT BR44_TVP17930/ R3 2012 isolate serum_44, complete genomeAIQ84241 3387 4 Brazil UTR5CMENS1NS Mar. 23, 2012 Dengue virus 42ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UT BR47_TVP17933/ R3 2012isolate serum_47, complete genome AIQ84240 3387 4 Brazil UTR5CMENS1NSMar. 21, 2012 Dengue virus 4 2ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UTBR48_TVP17934/ R3 2012 isolate serum_48, complete genome AIQ84239 3387 4Brazil UTR5CMENS1NS Mar. 12, 2012 Dengue virus 4 2ANS2BNS3NS4 strainDENV-4/MT/ A2KNS4BNS5UT BR50_TVP18148/ R3 2012 isolate serum_50,complete genome AIQ84238 3387 4 Brazil UTR5CMENS1NS Mar. 20, 2012 Denguevirus 4 2ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UT BR52_TVP17938/ R32012 isolate serum_52, complete genome AIQ84237 3387 4 BrazilUTR5CMENS1NS Mar. 14, 2012 Dengue virus 4 2ANS2BNS3NS4 strain DENV-4/MT/A2KNS4BNS5UT BR53_TVP17939/ R3 2012 isolate serum_53, complete genomeAIQ84236 3387 4 Brazil UTR5CMENS1NS Mar. 14, 2012 Dengue virus 42ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UT BR55_TVP17941/ R3 2012isolate serum_55, complete genome AIQ84235 3387 4 Brazil UTR5CMENS1NSMar. 14, 2012 Dengue virus 4 2ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UTBR60_TVP17946/ R3 2012 isolate serum_60, complete genome AIQ84234 3387 4Brazil UTR5CMENS1NS Apr. 19, 2012 Dengue virus 4 2ANS2BNS3NS4 strainDENV-4/MT/ A2KNS4BNS5UT BR73_TVP17951/ R3 2012 isolate serum_73,complete genome AIQ84233 3387 4 Brazil UTR5CMENS1NS Apr. 19, 2012 Denguevirus 4 2ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UT BR76_TVP17953/ R32012 isolate serum_76, complete genome AIQ84232 3387 4 BrazilUTR5CMENS1NS Feb. 3, 2012 Dengue virus 4 2ANS2BNS3NS4 strain DENV-4/MT/A2KNS4BNS5UT BR84_TVP17961/ R3 2012 isolate serum_84, complete genomeAIQ84221 3387 4 Brazil UTR5CMENS1NS Apr. 23, 2012 Dengue virus 42ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UT BR8_TVP17894/ R3 2012isolate serum_8, complete genome AIQ84231 3387 4 Brazil UTR5CMENS1NSFeb. 3, 2012 Dengue virus 4 2ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UTBR91_TVP17968/ R3 2012 isolate serum_91, complete genome AIQ84230 3387 4Brazil UTR5CMENS1NS Feb. 29, 2012 Dengue virus 4 2ANS2BNS3NS4 strainDENV-4/MT/ A2KNS4BNS5UT BR92_TVP17969/ R3 2012 isolate serum_92,complete genome AIQ84229 3387 4 Brazil UTR5CMENS1NS Feb. 16, 2012 Denguevirus 4 2ANS2BNS3NS4 strain DENV-4/MT/ A2KNS4BNS5UT BR94_TVP17971/ R32012 isolate serum_94, complete genome AIQ84222 3387 4 BrazilUTR5CMENS1NS Apr. 18, 2012 Dengue virus 4 2ANS2BNS3NS4 strain DENV-4/MT/A2KNS4BNS5UT BR9_TVP17895/ R3 2012 isolate serum_9, complete genomeAEW50182 3387 4 Brazil UTR5CMENS1NS Mar. 26, 1982 Dengue virus 42ANS2BNS3NS4 strain H402276, A2KNS4BNS5 complete genome AFX65866 3387 4Brazil UTR5CMENS1NS Jul. 17, 2010 Dengue virus 4 2ANS2BNS3NS4 strainH772846, A2KNS4BNS5UT complete genome R3 AFX65867 3387 4 BrazilUTR5CMENS1NS Jul. 18, 2010 Dengue virus 4 2ANS2BNS3NS4 strain H772852,A2KNS4BNS5UT complete genome R3 AEW50183 3387 4 Brazil UTR5CMENS1NS Jul.21, 2010 Dengue virus 4 2ANS2BNS3NS4 strain H772854, A2KNS4BNS5 completegenome AFX65868 3387 4 Brazil UTR5CMENS1NS Aug. 20, 2010 Dengue virus 42ANS2BNS3NS4 strain H773583, A2KNS4BNS5UT complete genome R3 AFX658693387 4 Brazil UTR5CMENS1NS Aug. 24, 2010 Dengue virus 4 2ANS2BNS3NS4strain H774846, A2KNS4BNS5UT complete genome R3 AFX65870 3387 4 BrazilUTR5CMENS1NS Nov. 10, 2010 Dengue virus 4 2ANS2BNS3NS4 strain H775222,A2KNS4BNS5UT complete genome R3 AFX65871 3387 4 Brazil UTR5CMENS1NS Jan.12, 2011 Dengue virus 4 2ANS2BNS3NS4 strain H778494, A2KNS4BNS5UTcomplete genome R3 AFX65872 3387 4 Brazil UTR5CMENS1NS Jan. 11, 2011Dengue virus 4 2ANS2BNS3NS4 strain H778504, A2KNS4BNS5UT complete genomeR3 AFX65873 3387 4 Brazil UTR5CMENS1NS Jan. 20, 2011 Dengue virus 42ANS2BNS3NS4 strain H778887, A2KNS4BNS5UT complete genome R3 AFX658743387 4 Brazil UTR5CMENS1NS Jan. 14, 2011 Dengue virus 4 2ANS2BNS3NS4strain H779228, A2KNS4BNS5UT complete genome R3 AFX65875 3387 4 BrazilUTR5CMENS1NS Jan. 24, 2011 Dengue virus 4 2ANS2BNS3NS4 strain H779652,A2KNS4BNS5UT complete genome R3 AFX65876 3387 4 Brazil UTR5CMENS1NS Nov.29, 2010 Dengue virus 4 2ANS2BNS3NS4 strain H780090, A2KNS4BNS5UTcomplete genome R3 AFX65877 3387 4 Brazil UTR5CMENS1NS Nov. 21, 2010Dengue virus 4 2ANS2BNS3NS4 strain H780120, A2KNS4BNS5UT complete genomeR3 AFX65878 3387 4 Brazil UTR5CMENS1NS Jan. 29, 2011 Dengue virus 42ANS2BNS3NS4 strain H780556, A2KNS4BNS5UT complete genome R3 AFX658793387 4 Brazil UTR5CMENS1NS Jan. 29, 2011 Dengue virus 4 2ANS2BNS3NS4strain H780563, A2KNS4BNS5UT complete genome R3 AFX65880 3387 4 BrazilUTR5CMENS1NS Jan. 13, 2011 Dengue virus 4 2ANS2BNS3NS4 strain H780571,A2KNS4BNS5UT complete genome R3 AFX65881 3387 4 Brazil UTR5CMENS1NS Mar.18, 2011 Dengue virus 4 2ANS2BNS3NS4 strain H781363, A2KNS4BNS5UTcomplete genome R3 AIK23224 3391 2 Cuba CMENS1NS2ANS 1981 Dengue virus 22BNS3NS4A2KN isolate S4BNS5 Cuba_A115_1981 polyprotein gene, completecds AIK23223 3391 2 Cuba CMENS1NS2ANS 1981 Dengue virus 2 2BNS3NS4A2KNisolate S4BNS5 Cuba_A132_1981 polyprotein gene, complete cds AIK232223391 2 Cuba CMENS1NS2ANS 1981 Dengue virus 2 2BNS3NS4A2KN isolate S4BNS5Cuba_A15_1981 polyprotein gene, complete cds AIK23225 3391 2 CubaCMENS1NS2ANS 1981 Dengue virus 2 2BNS3NS4A2KN isolate S4BNS5Cuba_A169_1981 polyprotein gene, complete cds AIK23226 3391 2 CubaCMENS1NS2ANS 1981 Dengue virus 2 2BNS3NS4A2KN isolate S4BNS5Cuba_A35_1981 polyprotein gene, complete cds AAW31409 3391 2 CubaUTR5CMENS1NS Dengue virus type 2ANS2BNS3NS4 2 strain A2KNS4BNS5UTCuba115/97, R3 complete genome AAW31407 3391 2 Cuba UTR5CMENS1NS Denguevirus type 2ANS2BNS3NS4 2 strain A2KNS4BNS5UT Cuba13/97, R3 completegenome AAW31411 3391 2 Cuba UTR5CMENS1NS Dengue virus type 2ANS2BNS3NS42 strain A2KNS4BNS5UT Cuba165/97, R3 complete genome AAW31412 3391 2Cuba UTR5CMENS1NS 1997 Dengue virus type 2ANS2BNS3NS4 2 strainA2KNS4BNS5UT Cuba205/97, R3 complete genome AAW31408 3391 2 CubaUTR5CMENS1NS Dengue virus type 2ANS2BNS3NS4 2 strain A2KNS4BNS5UTCuba58/97, R3 complete genome AAW31410 3391 2 Cuba UTR5CMENS1NS 1997Dengue virus type 2ANS2BNS3NS4 2 strain A2KNS4BNS5UT Cuba89/97, R3complete genome AFJ91714 3392 1 USA UTR5CMENS1NS 2010, October Denguevirus 1 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 1/BOL-KW010, R3 completegenome ACA48834 3392 1 USA UTR5CMENS1NS 1998 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V1162/1998, complete genomeACJ04186 3392 1 USA UTR5CMENS1NS 1995 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V1734/1995, complete genomeACJ04190 3392 1 USA UTR5CMENS1NS 1998 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V1738/1998, complete genomeACH99678 3392 1 USA UTR5CMENS1NS 1998 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V1739/1998, complete genomeACH99679 3392 1 USA UTR5CMENS1NS 1998 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V1740/1998, complete genomeACJ04191 3392 1 USA UTR5CMENS1NS 1998 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V1741/1998, complete genomeACJ04192 3392 1 USA UTR5CMENS1NS 1998 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V1742/1998, complete genomeACH99680 3392 1 USA UTR5CMENS1NS 1995 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V1743/1995, complete genomeACH99681 3392 1 USA UTR5CMENS1NS 1995 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V1744/1995, complete genomeACJ04215 3392 1 USA UTR5CMENS1NS 1998 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2093/1998, complete genomeACJ04216 3392 1 USA UTR5CMENS1NS 1995 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2094/1995, complete genomeACJ04217 3392 1 USA UTR5CMENS1NS 1994 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2095/1994, complete genomeACL99012 3392 1 USA UTR5CMENS1NS 1993 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2096/1993, complete genomeACL99013 3392 1 USA UTR5CMENS1NS 1986 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2097/1986, complete genomeACJ04221 3392 1 USA UTR5CMENS1NS 1994 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2127/1994, complete genomeACJ04222 3392 1 USA UTR5CMENS1NS 1995 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2128/1995, complete genomeACJ04223 3392 1 USA UTR5CMENS1NS 1995 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2129/1995, complete genomeACL99002 3392 1 USA UTR5CMENS1NS 1995 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2130/1995, complete genomeACJ04224 3392 1 USA UTR5CMENS1NS 1996 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2131/1996, complete genomeACJ04225 3392 1 USA UTR5CMENS1NS 1993 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2132/1993, complete genomeACJ04226 3392 1 USA UTR5CMENS1NS 1993 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2133/1993, complete genomeACJ04227 3392 1 USA UTR5CMENS1NS 1993 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2134/1993, complete genomeACL99003 3392 1 USA UTR5CMENS1NS 1992 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2135/1992, complete genomeACJ04228 3392 1 USA UTR5CMENS1NS 1992 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2136/1992, complete genomeACJ04229 3392 1 USA UTR5CMENS1NS 1992 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2137/1992, complete genomeACK28188 3392 1 USA UTR5CMENS1NS 1996 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2138/1996, complete genomeACJ04230 3392 1 USA UTR5CMENS1NS 1996 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2139/1996, complete genomeACJ04231 3392 1 USA UTR5CMENS1NS 1996 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2140/1996, complete genomeACK28189 3392 1 USA UTR5CMENS1NS 1987 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2142/1987, complete genomeACJ04232 3392 1 USA UTR5CMENS1NS 1987 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V2143/1987, complete genomeACA48858 3392 1 USA UTR5CMENS1NS 2006 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V852/2006, complete genomeACA48859 3392 1 USA UTR5CMENS1NS 1998 Dengue virus 1 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 1/US/BID- R3 V853/1998, complete genomeACF49259 3392 1 USA UTR5CMENS1NS 1944 Dengue virus 1 2ANS2BNS3NS4isolate A2KNS4BNS5UT US/Hawaii/1944, R3 complete genome AIU47321 3392 1USA UTR5CMENS1NS 1944 Dengue virus 1 2ANS2BNS3NS4 strain Hawaii,A2KNS4BNS5UT complete genome R3 ACA48811 3391 2 USA UTR5CMENS1NS 2006Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3V1031/2006, complete genome ACA48812 3391 2 USA UTR5CMENS1NS 1998 Denguevirus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1032/1998,complete genome ACA48813 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1033/1998,complete genome ACA48814 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1034/1998,complete genome ACA48815 3391 2 USA UTR5CMENS1NS 2006 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1035/2006,complete genome ACA48816 3391 2 USA UTR5CMENS1NS 2006 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1036/2006,complete genome ACA48817 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1038/1998,complete genome ACA48818 3391 2 USA UTR5CMENS1NS 2006 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1039/2006,complete genome ACA48819 3391 2 USA UTR5CMENS1NS 2006 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1040/2006,complete genome ACA48820 3391 2 USA UTR5CMENS1NS 2006 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1041/2006,complete genome ACA48821 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1042/1998,complete genome ACA48823 3391 2 USA UTR5CMENS1NS 2005 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1045/2005,complete genome ACA58330 3391 2 USA UTR5CMENS1NS 2004 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1046/2004,complete genome ACA48824 3391 2 USA UTR5CMENS1NS 1999 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1048/1999,complete genome ACA48827 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1052/1998,complete genome ACA48828 3391 2 USA UTR5CMENS1NS 1996 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1054/1996,complete genome ACB29511 3391 2 USA UTR5CMENS1NS 1996 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1055/1996,complete genome ACA58331 3391 2 USA UTR5CMENS1NS 1994 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1057/1994,complete genome ACA58332 3391 2 USA UTR5CMENS1NS 1994 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1058/1994,complete genome ACA48829 3391 2 USA UTR5CMENS1NS 1989 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1060/1989,complete genome ACD13309 3391 2 USA UTR5CMENS1NS 1989 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1061/1989,complete genome ACA48832 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1084/1998,complete genome ACA58337 3391 2 USA UTR5CMENS1NS 1994 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1085/1994,complete genome ACA58338 3391 2 USA UTR5CMENS1NS 1991 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1087/1991,complete genome ACB29512 3391 2 USA UTR5CMENS1NS 1986 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1163/1986,complete genome ACA48835 3391 2 USA UTR5CMENS1NS 1986 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1164/1986,complete genome ACA48836 3391 2 USA UTR5CMENS1NS 1987 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1165/1987,complete genome ACA48837 3391 2 USA UTR5CMENS1NS 1987 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1166/1987,complete genome ACA48838 3391 2 USA UTR5CMENS1NS 1987 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1167/1987,complete genome ACA48839 3391 2 USA UTR5CMENS1NS 1987 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1168/1987,complete genome ACA48840 3391 2 USA UTR5CMENS1NS 1987 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1169/1987,complete genome ACA48841 3391 2 USA UTR5CMENS1NS 1987 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1170/1987,complete genome ACA48842 3391 2 USA UTR5CMENS1NS 1987 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1171/1987,complete genome ACA48843 3391 2 USA UTR5CMENS1NS 1987 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1172/1987,complete genome ACA48844 3391 2 USA UTR5CMENS1NS 1987 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1174/1987,complete genome ACA48845 3391 2 USA UTR5CMENS1NS 1988 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1175/1988,complete genome ACA48846 3391 2 USA UTR5CMENS1NS 1988 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1176/1988,complete genome ACA48847 3391 2 USA UTR5CMENS1NS 1989 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1177/1989,complete genome ACA48848 3391 2 USA UTR5CMENS1NS 1989 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1178/1989,complete genome ACA48849 3391 2 USA UTR5CMENS1NS 1989 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1179/1989,complete genome ACA48850 3391 2 USA UTR5CMENS1NS 1989 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1180/1989,complete genome ACA48851 3391 2 USA UTR5CMENS1NS 1989 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1181/1989,complete genome ACA48852 3391 2 USA UTR5CMENS1NS 1989 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1182/1989,complete genome ACA48853 3391 2 USA UTR5CMENS1NS 1990 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1183/1990,complete genome ACA48854 3391 2 USA UTR5CMENS1NS 1990 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1188/1990,complete genome ACA48855 3391 2 USA UTR5CMENS1NS 1990 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1189/1990,complete genome ACB29513 3391 2 USA UTR5CMENS1NS 1993 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1356/1993,complete genome ACA48856 3391 2 USA UTR5CMENS1NS 1993 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1360/1993,complete genome ACB29514 3391 2 USA UTR5CMENS1NS 1995 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1367/1995,complete genome ACB29515 3391 2 USA UTR5CMENS1NS 1995 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1368/1995,complete genome ACD13310 3391 2 USA UTR5CMENS1NS 1995 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1372/1995,complete genome ACB29516 3391 2 USA UTR5CMENS1NS 1995 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1373/1995,complete genome ACB29517 3391 2 USA UTR5CMENS1NS 1996 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1376/1996,complete genome ACB87126 3391 2 USA UTR5CMENS1NS 1996 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1378/1996,complete genome ACB29518 3391 2 USA UTR5CMENS1NS 1996 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1383/1996,complete genome ACB87127 3391 2 USA UTR5CMENS1NS 1996 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1385/1996,complete genome ACD13396 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1387/1998,complete genome ACD13311 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1388/1998,complete genome ACB29519 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1392/1998,complete genome ACB29520 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1393/1998,complete genome ACB87128 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1394/1998,complete genome ACB29521 3391 2 USA UTR5CMENS1NS 1997 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1395/1997,complete genome ACB29522 3391 2 USA UTR5CMENS1NS 1997 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1396/1997,complete genome ACB29523 3391 2 USA UTR5CMENS1NS 1997 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1397/1997,complete genome ACB29524 3391 2 USA UTR5CMENS1NS 1997 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1398/1997,complete genome ACB29525 3391 2 USA UTR5CMENS1NS 1997 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1399/1997,complete genome ACB29526 3391 2 USA UTR5CMENS1NS 1997 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1401/1997,complete genome ACB29527 3391 2 USA UTR5CMENS1NS 1997 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1404/1997,complete genome ACB29528 3391 2 USA UTR5CMENS1NS 1997 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1409/1997,complete genome ACB87129 3391 2 USA UTR5CMENS1NS 2007 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1410/2007,complete genome ACB87130 3391 2 USA UTR5CMENS1NS 2007 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1411/2007,complete genome ACB87131 3391 2 USA UTR5CMENS1NS 2007 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1412/2007,complete genome ACB87132 3391 2 USA UTR5CMENS1NS 2007 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1413/2007,complete genome ACD13348 3391 2 USA UTR5CMENS1NS 1996 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1424/1996,complete genome ACD13349 3391 2 USA UTR5CMENS1NS 1999 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1425/1999,complete genome ACD13350 3391 2 USA UTR5CMENS1NS 1999 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1426/1999,complete genome ACD13351 3391 2 USA UTR5CMENS1NS 1999 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1427/1999,complete genome ACD13352 3391 2 USA UTR5CMENS1NS 1999 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1428/1999,complete genome ACD13353 3391 2 USA UTR5CMENS1NS 2004 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/B ID- R3 V1431/2004,complete genome ACD13354 3391 2 USA UTR5CMENS1NS 2004 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1432/2004,complete genome ACD13397 3391 2 USA UTR5CMENS1NS 2004 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1434/2004,complete genome ACD13398 3391 2 USA UTR5CMENS1NS 2004 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1435/2004,complete genome ACD13399 3391 2 USA UTR5CMENS1NS 2004 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1436/2004,complete genome ACD13400 3391 2 USA UTR5CMENS1NS 2005 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1439/2005,complete genome ACE63530 3391 2 USA UTR5CMENS1NS 2005 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1440/2005,complete genome ACD13401 3391 2 USA UTR5CMENS1NS 2005 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1441/2005,complete genome ACE63543 3391 2 USA UTR5CMENS1NS 2005 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1442/2005,complete genome ACD13406 3391 2 USA UTR5CMENS1NS 2000 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1461/2000,complete genome ACD13407 3391 2 USA UTR5CMENS1NS 2000 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1462/2000,complete genome ACD13408 3391 2 USA UTR5CMENS1NS 2000 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1463/2000,complete genome ACD13409 3391 2 USA UTR5CMENS1NS 2000 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1464/2000,complete genome ACD13411 3391 2 USA UTR5CMENS1NS 2001 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1467/2001,complete genome ACD13412 3391 2 USA UTR5CMENS1NS 2001 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1468/2001,complete genome ACD13413 3391 2 USA UTR5CMENS1NS 2001 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1469/2001,complete genome ACD13414 3391 2 USA UTR5CMENS1NS 2001 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1470/2001,complete genome ACD13415 3391 2 USA UTR5CMENS1NS 2001 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1471/2001,complete genome ACD13416 3391 2 USA UTR5CMENS1NS 2001 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1472/2001,complete genome ACD13395 3391 2 USA UTR5CMENS1NS 2003 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1482/2003,complete genome ACD13419 3391 2 USA UTR5CMENS1NS 2003 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1483/2003,complete genome ACD13420 3391 2 USA UTR5CMENS1NS 2003 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1484/2003,complete genome ACD13421 3391 2 USA UTR5CMENS1NS 2003 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1486/2003,complete genome ACD13422 3391 2 USA UTR5CMENS1NS 2003 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1487/2003,complete genome ACD13424 3391 2 USA UTR5CMENS1NS 2003 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1492/2003,complete genome ACD13425 3391 2 USA UTR5CMENS1NS 2003 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1493/2003,complete genome ACD13426 3391 2 USA UTR5CMENS1NS 2004 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1494/2004,complete genome ACD13427 3391 2 USA UTR5CMENS1NS 2004 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1495/2004,complete genome ACD13428 3391 2 USA UTR5CMENS1NS 2004 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1496/2004,complete genome ACD13429 3391 2 USA UTR5CMENS1NS 2005 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V1497/2005,complete genome AEH59341 3390 2 USA CMENS1NS2ANS 2009 Dengue virus 22BNS3NS4A2KN isolate DENV- S4BNS5 2/US/BID- V4824/2009, complete genomeAEH59346 3391 2 USA UTR5CMENS1NS 2006 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5 2/US/BID- V5411/2006, complete genome AEH593453391 2 USA UTR5CMENS1NS 2007 Dengue virus 2 2ANS2BNS3NS4 isolate DENV-A2KNS4BNS5 2/US/BID- V5412/2007, complete genome ACA58343 3391 2 USAUTR5CMENS1NS 2006 Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT2/US/BID- R3 V585/2006, complete genome ACA48986 3391 2 USA UTR5CMENS1NS2006 Dengue virus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3V587/2006, complete genome ACA48987 3391 2 USA UTR5CMENS1NS 2006 Denguevirus 2 2ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V588/2006,complete genome ACA48988 3391 2 USA UTR5CMENS1NS 2006 Dengue virus 22ANS2BNS3NS4 isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V589/2006, completegenome ACA48989 3391 2 USA UTR5CMENS1NS 2002 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V591/2002, complete genomeACA48990 3391 2 USA UTR5CMENS1NS 2002 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V592/2002, complete genomeACA48991 3391 2 USA UTR5CMENS1NS 2005 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V593/2005, complete genomeACA48992 3391 2 USA UTR5CMENS1NS 2006 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V594/2006, complete genomeACA48993 3391 2 USA UTR5CMENS1NS 2006 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V595/2006, complete genomeACA48994 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V596/1998, complete genomeACA48995 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V597/1998, complete genomeACA48996 3391 2 USA UTR5CMENS1NS 1999 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V598/1999, complete genomeACA48997 3391 2 USA UTR5CMENS1NS 1999 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V599/1999, complete genomeACA48998 3391 2 USA UTR5CMENS1NS 2005 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V600/2005, complete genomeACA48999 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V675/1998, complete genomeACA49000 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V676/1998, complete genomeACA49001 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V677/1998, complete genomeACA49002 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V678/1998, complete genomeACA49003 3391 2 USA UTR5CMENS1NS 1994 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V679/1994, complete genomeACA49004 3391 2 USA UTR5CMENS1NS 1994 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V680/1994, complete genomeACA49005 3391 2 USA UTR5CMENS1NS 1998 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V681/1998, complete genomeACA49006 3391 2 USA UTR5CMENS1NS 1994 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V682/1994, complete genomeACA49007 3391 2 USA UTR5CMENS1NS 1994 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V683/1994, complete genomeACA49008 3391 2 USA UTR5CMENS1NS 1994 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V684/1994, complete genomeACA49009 3391 2 USA UTR5CMENS1NS 1988 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V685/1988, complete genomeACA49010 3391 2 USA UTR5CMENS1NS 1989 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V686/1989, complete genomeACA49011 3391 2 USA UTR5CMENS1NS 1989 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V687/1989, complete genomeACA49012 3391 2 USA UTR5CMENS1NS 1989 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V688/1989, complete genomeACA49013 3391 2 USA UTR5CMENS1NS 1989 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V689/1989, complete genomeACA49014 3391 2 USA UTR5CMENS1NS 1988 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V690/1988, complete genomeACA48857 3391 2 USA UTR5CMENS1NS 1990 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V851/1990, complete genomeACA48860 3391 2 USA UTR5CMENS1NS 2001 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V854/2001, complete genomeACA48861 3391 2 USA UTR5CMENS1NS 1992 Dengue virus 2 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 2/US/BID- R3 V855/1992, complete genomeACA48822 3390 3 USA UTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1043/2006, complete genomeACA58329 3390 3 USA UTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1044/2006, complete genomeACA48825 3390 3 USA UTR5CMENS1NS 1998 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1049/1998, complete genomeACA48826 3390 3 USA UTR5CMENS1NS 1998 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1050/1998, complete genomeACA48830 3390 3 USA UTR5CMENS1NS 1998 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1075/1998, complete genomeACA58333 3390 3 USA UTR5CMENS1NS 1999 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1076/1999, complete genomeACA58334 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1077/2000, complete genomeACA48831 3390 3 USA UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1078/2003, complete genomeACA58335 3390 3 USA UTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1079/2006, complete genomeACA58336 3390 3 USA UTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1080/2006, complete genomeACA48833 3390 3 USA UTR5CMENS1NS 1998 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1088/1998, complete genomeACA58339 3390 3 USA UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1089/2003, complete genomeACA58340 3390 3 USA UTR5CMENS1NS 1998 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1090/1998, complete genomeACA58341 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1091/2004, complete genomeACA58342 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1092/2004, complete genomeACB87133 3390 3 USA UTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1415/2007, complete genomeACB87134 3390 3 USA UTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1416/2007, complete genomeACB87135 3390 3 USA UTR5CMENS1NS 2007 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1417/2007, complete genomeACD13402 3390 3 USA UTR5CMENS1NS 1998 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1447/1998, complete genomeACE63531 3390 3 USA UTR5CMENS1NS 1998 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1448/1998, complete genomeACE63532 3390 3 USA UTR5CMENS1NS 1998 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1449/1998, complete genomeACH99660 3390 3 USA UTR5CMENS1NS 1998 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1450/1998, complete genomeACE63544 3390 3 USA UTR5CMENS1NS 1999 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1451/1999, complete genomeACE63545 3390 3 USA UTR5CMENS1NS 1999 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1452/1999, complete genomeACE63533 3390 3 USA UTR5CMENS1NS 1999 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1453/1999, complete genomeACE63534 3390 3 USA UTR5CMENS1NS 1999 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1454/1999, complete genomeACD13403 3390 3 USA UTR5CMENS1NS 1999 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1455/1999, complete genomeACD13405 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1460/2000, complete genomeACE63528 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1465/2000, complete genomeACD13410 3390 3 USA UTR5CMENS1NS 1999 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1466/1999, complete genomeACD13417 3391 3 USA UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1473/2002, complete genomeACD13418 3390 3 USA UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1475/2002, complete genomeACD13392 3390 3 USA UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1476/2002, complete genomeACH61690 3390 3 USA UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1477/2002, complete genomeACJ04182 3390 3 USA UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1478/2002, complete genomeACD13393 3390 3 USA UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1480/2003, complete genomeACD13394 3390 3 USA UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1481/2003, complete genomeACE63529 3390 3 USA UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1490/2003, complete genomeACD13423 3390 3 USA UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1491/2003, complete genomeACH99651 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1604/2004, complete genomeACO06143 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1605/2004, complete genomeACH61715 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1606/2004, complete genomeACH61716 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1607/2004, complete genomeACH61717 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1608/2004, complete genomeACH61718 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1609/2004, complete genomeACH61719 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1610/2004, complete genomeACO06144 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1611/2004, complete genomeACH61720 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1612/2004, complete genomeACH61721 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1613/2004, complete genomeACH99652 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1614/2004, complete genomeACJ04178 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1615/2004, complete genomeACH99653 3390 3 USA UTR5CMENS1NS 2004 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1616/2004, complete genomeACH99654 3390 3 USA UTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1617/2005, complete genomeACH99655 3390 3 USA UTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1618/2005, complete genomeACH99656 3390 3 USA UTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1619/2005, complete genomeACH99657 3390 3 USA UTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1620/2005, complete genomeACH99658 3390 3 USA UTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1621/2005, complete genomeACH99665 3390 3 USA UTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1622/2005, complete genomeACH99666 3390 3 USA UTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1623/2005, complete genomeACH99667 3390 3 USA UTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1624/2005, complete genomeACH99668 3390 3 USA UTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1625/2005, complete genomeACH99669 3390 3 USA UTR5CMENS1NS 2005 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1626/2005, complete genomeACJ04183 3390 3 USA UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1729/2003, complete genomeACJ04184 3390 3 USA UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1730/2003, complete genomeACH99676 3390 3 USA UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1731/2003, complete genomeACJ04185 3390 3 USA UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1732/2002, complete genomeACH99677 3390 3 USA UTR5CMENS1NS 1999 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1733/1999, complete genomeACJ04187 3390 3 USA UTR5CMENS1NS 1999 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1735/1999, complete genomeACJ04188 3390 3 USA UTR5CMENS1NS 1999 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1736/1999, complete genomeACJ04189 3390 3 USA UTR5CMENS1NS 1999 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V1737/1999, complete genomeACL98985 3390 3 USA UTR5CMENS1NS 1999 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2098/1999, complete genomeACL98986 3390 3 USA UTR5CMENS1NS 1998 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2099/1998, complete genomeACL99014 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2100/2000, complete genomeACL98987 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2103/2000, complete genomeACJ04218 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2104/2000, complete genomeACJ04219 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2105/2000, complete genomeACL98988 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2106/2000, complete genomeACL98989 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2107/2000, complete genomeACL98990 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2108/2000, complete genomeACL98991 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2110/2000, complete genomeACL98992 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2111/2000, complete genomeACL98993 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2112/2000, complete genomeACL98994 3390 3 USA UTR5CMENS1NS 2000 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2113/2000, complete genomeACL98995 3390 3 USA UTR5CMENS1NS 2001 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2114/2001, complete genomeACL98996 3390 3 USA UTR5CMENS1NS 2001 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2115/2001, complete genomeACL98997 3390 3 USA UTR5CMENS1NS 2001 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2117/2001, complete genomeACL98998 3390 3 USA UTR5CMENS1NS 2001 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2118/2001, complete genomeACL98999 3390 3 USA UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2119/2002, complete genomeACJ04220 3390 3 USA UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2120/2002, complete genomeACL99000 3390 3 USA UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2122/2002, complete genomeACK28187 3390 3 USA UTR5CMENS1NS 2002 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2123/2002, complete genomeACL99001 3390 3 USA UTR5CMENS1NS 2006 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V2126/2006, complete genomeACA48862 3390 3 USA UTR5CMENS1NS 2003 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V858/2003, complete genomeACA48863 3390 3 USA UTR5CMENS1NS 1998 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/US/BID- R3 V859/1998, complete genomeAFZ40124 3390 3 USA UTR5CMENS1NS 1963 Dengue virus 3 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 3/USA/633798/ R3 1963, complete genomeACH61714 3387 4 USA UTR5CMENS1NS 1998 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V1082/1998, complete genomeACH61687 3387 4 USA UTR5CMENS1NS 1986 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V1083/1986, complete genomeACH61688 3387 4 USA UTR5CMENS1NS 1998 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V1093/1998, complete genomeACH61689 3387 4 USA UTR5CMENS1NS 1998 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V1094/1998, complete genomeACS32012 3387 4 USA UTR5CMENS1NS 1994 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2429/1994, complete genomeACS32013 3387 4 USA UTR5CMENS1NS 1994 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2430/1994, complete genomeACS32014 3387 4 USA UTR5CMENS1NS 1995 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2431/1995, complete genomeACS32037 3387 4 USA UTR5CMENS1NS 1995 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2432/1995, complete genomeACO06140 3387 4 USA UTR5CMENS1NS 1995 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2433/1995, complete genomeACO06145 3387 4 USA UTR5CMENS1NS 1995 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2434/1995, complete genomeACS32015 3387 4 USA UTR5CMENS1NS 1996 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2435/1996, complete genomeACS32016 3387 4 USA UTR5CMENS1NS 1996 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2436/1996, complete genomeACS32017 3387 4 USA UTR5CMENS1NS 1996 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2437/1996, complete genomeACS32018 3387 4 USA UTR5CMENS1NS 1996 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2438/1996, complete genomeACS32019 3387 4 USA UTR5CMENS1NS 1996 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2439/1996, complete genomeACO06146 3387 4 USA UTR5CMENS1NS 1996 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2440/1996, complete genomeACQ44402 3387 4 USA UTR5CMENS1NS 1998 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2441/1998, complete genomeACQ44403 3387 4 USA UTR5CMENS1NS 1998 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2442/1998, complete genomeACO06147 3387 4 USA UTR5CMENS1NS 1998 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2443/1998, complete genomeACQ44404 3387 4 USA UTR5CMENS1NS 1998 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2444/1998, complete genomeACQ44405 3387 4 USA UTR5CMENS1NS 1998 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2445/1998, complete genomeACQ44406 3387 4 USA UTR5CMENS1NS 1999 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2446/1999, complete genomeACQ44407 3387 4 USA UTR5CMENS1NS 1999 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2447/1999, complete genomeACQ44408 3387 4 USA UTR5CMENS1NS 1999 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V2448/1999, complete genomeACJ04171 3387 4 USA UTR5CMENS1NS 1994 Dengue virus 4 2ANS2BNS3NS4isolate DENV- A2KNS4BNS5UT 4/US/BID- R3 V860/1994, complete genome

TABLE 36 DENV POLYPEPTIDE SEQUENCES SEQ ID NO: Accession No. Sequence171 gi|158348409|ref| MNNQRKKTGRPSFNMLKRARNRVSTVSQLAKRFSKGLLNP_722466.2| SGQGPMKLVMAFIAFLRFLAIPPTAGILARWGSFKKNGAI capsid proteinKVLRGFKKEISNMLNIMNRRKR [Dengue virus 1] 172 gi|164654862|ref|MNNQRKKTGKPSINMLKRVRNRVSTGSQLAKRFSKGLL YP_001531164.2|NGQGPMKLVMAFIAFLRFLAIPPTAGVLARWGTFKKSGA Capsid proteinIKVLKGFKKEISNMLSIINQRKK [Dengue virus 3] 173 gi|159024809|ref|MNNQRKKAKNTPFNMLKRERNRVSTVQQLTKRFSLGM NP_739591.2|LQGRGPLKLFMALVAFLRFLTIPPTAGILKRWGTIKKSKA Capsid proteinINVLRGFRKEIGRMLNILNRRRR [Dengue virus 2] 174 gi|158348408|ref|MNNQRKKTGRPSFNMLKRARNRVSTVSQLAKRFSKGLL NP_722457.2|SGQGPMKLVMAFIAFLRFLAIPPTAGILARWGSFKKNGAI anchored capsidKVLRGFKKEISNMLNIMNRRKRSVTMLLMLLPTALA protein [Dengue virus 1] 175gi|164654854|ref| MNNQRKKTGKPSINMLKRVRNRVSTGSQLAKRFSKGLL YP_001531165.2|NGQGPMKLVMAFIAFLRFLAIPPTAGVLARWGTFKKSGA Anchored capsidIKVLKGFKKEISNMLSIINQRKKTSLCLMMILPAALA protein [Dengue virus 3] 176gi|159024808|ref| MNNQRKKAKNTPFNMLKRERNRVSTVQQLTKRFSLGM NP_739581.2|LQGRGPLKLFMALVAFLRFLTIPPTAGILKRWGTIKKSKA Anchored capsidINVLRGFRKEIGRMLNILNRRRRSAGMIIMLIPTVMA protein [Dengue virus 2] 177gi|73671168|ref| MNQRKKVVRPPFNMLKRERNRVSTPQGLVKRFSTGLFS NP_740314.1|GKGPLRMVLAFITFLRVLSIPPTAGILKRWGQLKKNKAIK anchored capsidILIGFRKEIGRMLNILNGRKRSTITLLCLIPTVMA (anchC) protein [Dengue virus 4] 178gi|73671167|ref| MNQRKKVVRPPFNMLKRERNRVSTPQGLVKRFSTGLFS NP_740313.1|GKGPLRMVLAFITFLRVLSIPPTAGILKRWGQLKKNKAIK virion capsidILIGFRKEIGRMLNILNGRKR (virC) protein [Dengue virus 4] Envelopegi|164654853|ref| MRCVGVGNRDFVEGLSGATWVDVVLEHGGCVTTMAKN ProteinYP_001531168.2| KPTLDIELQKTEATQLATLRKLCIEGKITNITTDSRCPTQG 179 EnvelopeEAVLPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVT protein [DengueCAKFQCLEPIEGKVVQYENLKYTVIITVHTGDQHQVGNE virus 3]TQGVTAEITPQASTTEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPWASGATTETPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEIQNSGGTSIFAGHLKCRLKMDKLELKGMSYAMCTNTFVLKKEVSETQHGTILIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAEPPFGESNIVIGIGDNALKINWYKKGSSIGKMFEATERGARRMAILGDTAWDFGSVGGVLNSLGKMVHQIFGSAYTALFSGVSWVMKIGIGVLLTWIGL NSKNTSMSFSCIAIGIITLYLGAVVQA 180gi|158828123|ref| MRCVGIGNRDFVEGLSGATWVDVVLEHGSCVTTMAKD NP_722460.2|KPTLDIELLKTEVTNPAVLRKLCIEAKISNTTTDSRCPTQG envelope proteinEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCA [Dengue virus 1]KFKCVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTTATITPQAPTSEIQLTDYGALTLDCSPRTGLDFNEMVLLTMKKKSWLVHKQWFLDLPLPWTSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEIQTSGTTTIFAGHLKCRLKMDKLILKGMSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGARRMAILGDTAWDFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGL NSRSTSLSMTCIAVGMVTLYLGVMVQA 181gi|159024812|ref| MRCIGMSNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNK NP_739583.2|PTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGE Envelope proteinPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCA [Dengue virus 2]MFRCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGM NSRSTSLSVTLVLVGIVTLYLGVMVQA 182tr|Q9IZI6|Q9IZI6_9FLAV MRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQ Envelopeprotein GKPTLDFELTKTTAKEVALLRTYCIEASISNITTATRCPTQ (Fragment)GEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVV OS = Dengue virusTCAKFSCSGKITGNLVRIENLEYTVVVTVHNGDTHAVGN 4 GN = E PE = 4DTSNHGVTAMITPRSPSVEVKLPDYGELTLDCEPRSGIDF SV = 1NEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCEVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGNSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGFLV LWIGTNSRNTSMAMTCIAVGGITLFLGF 183gi|73671170|ref| SVALTPHSGMGLETRAETWMSSEGAWKHAQRVESWILR NP_740316.1|NPGFALLAGFMAYMIGQTGIQRTVFFVLMMLVAPSYG membrane (M) protein [Dengue virus4] 184 gi|158828127|ref| SVALAPHVGMGLDTRTQTWMSAEGAWRQVEKVETWAYP_001531167.1| LRHPGFTILALFLAHYIGTSLTQKVVIFILLMLVTPSMT Membraneglycoprotein [Dengue virus 3] 185 gi|158828122|ref|SVALAPHVGLGLETRTETWMSSEGAWKQIQKVETWALR NP_722459.2|HPGFTVIALFLAHAIGTSITQKGIIFILLMLVTPSMA membrane glycoprotein [Denguevirus 1] 186 gi|159024811|ref| SVALVPHVGMGLETRTETWMSSEGAWKHVQRIETWILRNP_739592.2| HPGFTMMAAILAYTIGTTHFQRALIFILLTAVTPSMT Membrane glycoprotein[Dengue virus 2]

Example 36 OVA Multitope In Vitro Screening Assay Kinetic Analysis

As depicted in FIG. 35, antigen surface presentation is an inefficientprocess in the antigen presenting cells (APC). Peptides generated fromproteasome degradation of the antigens are presented with low efficiency(only 1 peptide of 10000 degraded molecules is actually presented).Thus, priming of CD8 T cells with APCs provides insufficient densitiesof surface peptide/MHC I complexes, resulting in weak respondersexhibiting impaired cytokine secretion and decreased memory pool. Toimprove DENV mRNA vaccines encoding concatemeric DENV antigens, an invitro assay was designed to test the linkers used to connect peptiderepeats, the number of peptide repeats, and sequences known to enhanceantigen presentation.

mRNA constructs encoding one or more OVA epitopes were configured withdifferent linker sequences, protease cleavage sites, and antigenpresentation enhancer sequences. Their respective sequences were asshown in Table 37. To perform the assay, 200 ng of each MC3-formulatedmRNA construct was transfected into JAWSII cells in a 24-well plate.Cells were isolated at 6, 24, and 48 hours post transfection and stainedwith fluorescently-labeled Anti-Mouse OVA257-264 (SIINFEKL) peptidebound to H-2Kb. Staining was analyzed on a LSRFortessa flow cytometer.Samples were run in triplicate. The Mean Fluorescent Intensity (MFI) foreach mRNA construct was measured and shown in FIG. 36. Constructs 2, 3,7, 9, and 10 showed enhanced surface presentation of the OVA epitope,indicating that the configurations of these constructs may be used forDENV mRNA vaccine. Construct 5 comprises a single OVA peptide and a KDELsequence that is known to prevent the secretion of a protein. Construct5 showed little surface antigen presentation because the secretion ofthe peptide was inhibited.

Example 37 Antibody Binding to DENV-1, 2, 3, and 4 prME Epitopes

DENV mRNA vaccines encoding concatemeric antigen epitopes were testedfor binding to antibodies known to recognize one or more DENV serotypes.To test antibody binding to the epitopes, 200 ng of DENV mRNA vaccinesencoding different Dengue prME epitopes were transfected into HeLa cellsin 24-well plates using the TransitIT-mRNA Transfection Kit (Mires Bio).The DENV mRNA vaccine constructs are shown in Table 34. Transfectionswere done in triplicate. After 24 hours, surface expression was detectedusing four different antibodies (10 μg/mL) followed by eithergoat-anti-human or anti-mouse AF700 secondary antibody (1/500). Signalgenerated from antibody binding are shown as Mean Fluorescent Intensity(MFI) (FIG. 37). Antibody D88 is known to recognize all 4 serotypes andbound to all antigen epitopes encoded by the DENV mRNA vaccineconstructs tested. Antibody 2D22 is known to recognize only DENV 2 andpreferentially bound to construct 21, which encodes DENV 2 antigenepitopes. Antibody 2D22 also showed weak binding to epitopes of otherDENV serotypes. Antibody 5J7 is known to recognize only DENV 3 and onlybound to antigen epitopes encoded by constructs 13, 19, and 20, whichencode DENV 3 antigen epitopes. Antibody 1-11 is known to bind stronglyto DENV 1 and 2, to bind weakly to DENV 3 and to bind little DENV 4.Antibody 1-11 bound to DENV 1, 2, and 3, and binding to DENV 3 antigenepitopes was stronger than binding to DENV 1 or 2 (FIG. 37).

TABLE 37 mRNA constructs that encode one or more OVA epitopes # ofAntigen Presentation SEQ ID Construct Peptides/Repeats Linker EnhancerSequence Amino acid Sequence NO: 1 8 OVA G/S — MLESIINFEKLTEGGGGS 187(8mer) GGGGSLESIINFEKLTEG Repeats GGGSGGGGSLESIINFEK (FlankingLTEGGGGSGGGGSLESII AA) NFEKLTEGGGGSGGGGSL ESIINFEKLTEGGGGSGGGGSLESIINFEKLTEGGG GSGGGGSLESIINFEKLT EGGGGSGGGGSLESIINF EKLTE 2 8 OVACathepsin B — MLESIINFEKLTEGFLGL 188 (8mer) Cleavage ESIINFEKLTEGFLGLESRepeats Site IINFEKLTEGFLGLESII (Flanking (GFLG) NFEKLTEGFLGLESIINF AA)EKLTEGFLGLESIINFEK LTEGFLGLESIINFEKLT EGFLGLESIINFEKLTE 3 8 OVA — HumanMHCI MRVTAPRTVLLLLSAALA 189 (8mer) Secretion LTETWALESIINFEKLTE RepeatsPeptide/Cytoplasmic LESIINFEKLTELESIIN (Flanking DomainFEKLTELESIINFEKLTE AA) LESIINFEKLTELESIIN FEKLTELESIINFEKLTELESIINFEKLTEGSIVGI VAGLAVLAVVVIGAVVAT VMCRRKSSGGKGGSYSQA ASSDSAQGSDVSLTA4 8 OVA Cathepsin B Human MHCI MRVTAPRTVLLLLSAALA 190 (8mer) CleavageSecretion LTETWALESIINFEKLTE Repeats Site Peptide/CytoplasmicGFLGLESIINFEKLTEGF (Flanking (GFLG) Domain LGLESIINFEKLTEGFLG AA)LESIINFEKLTEGFLGLE SIINFEKLTEGFLGLESI INFEKLTEGFLGLESIINFEKLTEGFLGLESIINFE KLTEGSIVGIVAGLAVLA VVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQG SDVSLTA 5 Single OVA — KDEL MSIINFEKLKDEL 191 6Single OVA — Human MHCI MRVTAPRTVLLLLSAALA 192 (Flanking SecretionLTETWALESIINFEKLTE AA) Peptide/Cytoplasmic GSIVGIVAGLAVLAVVVI DomainGAVVATVMCRRKSSGGKG GSYSQAASSDSAQGSDVS LTA 7 8 OVA Cathepsin B Murine IgKappa METDTLLLWVLLLWVPGS 193 (8mer) Cleavage Signal Peptide(Igκ)TGDSIINFEKLGFLGSII Repeats Site NFEKLGFLGSIINFEKLG (GFLG)FLGSIINFEKLGFLGSII NFEKLGFLGSIINFEKLG FLGSIINFEKLGFLGSII NFEKL 8 8 OVAG/S Human MHCI MRVTAPRTVLLLLSAALA 194 (8mer) Secretion LTETWLESIINFEKLTERepeats Peptide/Cytoplasmic GGGGSGGGGSLESIINFE (Flanking DomainKLTEGGGGSGGGGSLESI AA) INFEKLTEGGGGSGGGGS LESIINFEKLTEGGGGSGGGGSLESIINFEKLTEGG GGSGGGGSLESIINFEKL TEGGGGSGGGGSLESIINFEKLTEGGGGSGGGGSLE SIINFEKLTEGSIVGIVA GLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAAS SDSAQGSDVSLTA 9 8 OVA — — MLESIINFEKLTELESII 195(8mer) NFEKLTELESIINFEKLT Repeats ELESIINFEKLTELESII (FlankingNFEKLTELESIINFEKLT AA) ELESIINFEKLTELESII NFEKLTE 10 Single OVA — —MSIINFEKL 196 11 8 OVA Cathepsin B Murine Ig Kappa METDTLLLWVLLLWVPGS197 (8mer) Cleavage Signal Peptide(Igκ) TGDHPFTEDDAVDPNDSD Repeats Siteand PEST IDPESRSIINFEKLGFLG (GFLG) SIINFEKLGFLGSIINFE KLGFLGSIINFEKLGFLGSIINFEKLGFLGSIINFE KLGFLGSIINFEKLGFLG SIINFEKL 12 8 OVA Cathepsin BMurine MHC Class I MSIINFEKLGFLGSIINF 198 (8mer) Cleavage CytoplasmicDomain EKLGFLGSIINFEKLGFL Repeats Site (MITD) GSIINFEKLGFLGSIINF (GFLG)EKLGFLGSIINFEKLGFL GSIINFEKLGFLGSIINF EKLPPPSTVSNMIIIEVLIVLGAVINIGAMVAFVLK SKRKIGGKGGVYALAGGS NSIHGSALFLEAFKA

TABLE 38 DENV mRNA vaccine constructs tested for antibody binding or inchallenge studies Con- SEQ struct mRNA Name ID NO 13DEN3_prME_PaH881/88_AF349753.1 199 14 DEN1_prME_West_Pac_AY145121.1 20015 DEN1_prME_PUO-359_AAN32784.1 201 16 DEN4_prME_DHF_Patient_JN638571.1202 17 DEN4_prME_DENV4/CN/GZ29/2010_KP723482.1 203 18DEN4_prME_rDEN4_AF326825.1 204 19 DEN3_prME_L11439.1 205 20DEN3_prME_D3/Hu/TL129NIID/2005_AB214882 206 21DENV2_prME_Peru_IQT2913_1996 207 22 DENV2_prME_Thailand-168_1979 208 23DENV2_prME_Thailand_PUO-218_1980 209 (Sanofi strain) 24DEN2_D2Y98P_PRME80_Hs3_LSP 210 25 Non-H2Kb multitope 211 26 H2Kbmultitope 212

Example 38 DENV prME Challenge Study in Cynomolgus (cyno) Monkey Model

Shown in Table 39 is the design of DENV prME challenge study incynomolgus (cyno) money. Indicated DENV mRNA vaccine encoding prMEantigen epitopes, or vaccines thereof, are used to immunize cyno. Thevaccines are formulated in lipid nanoparticles (e.g., MC3 formulation)and administered to the cyno monkeys intramuscularly on day 0, 21, and42. Dosages of the vaccines are 250 μg or 5 μg per immunization. Inexperiments where a combination of different DENV mRNA vaccines areused, 250 μg or 5 μg of each mRNA vaccine is used. FLAG-tagged H10N8 fluvaccine is used as control at a dosage of 250 μg per immunization. Naïvecyno monkeys without immunization are also used as control. Cyno monkeysera are collected on days 20, 41, 62, and 92 post initial immunizationand used for serotype-specific neutralization assays.

Immunized cyno monkeys are challenged on day 63 post initialimmunization with indicated DENV viruses. Cyno monkey sera are collectedon days 62 (pre-challenge), 63-66, 68, 70, 72, 76, and 92 (end of life)to determine serum viral load.

TABLE 39 DENV prME Challenge Study Design in Cynomolgus (cyno) MonkeyGroup Vaccine n = 3 Vaccine Schedule Dosage/Route Challenge 1 Dengue 1Day 0, 21, 42 IM, LNP Challenge with prME 250 μg Dengue 1/03135 s.c 2(Construct IM, LNP (5log PFU) 15) 5 μg 3 Dengue 2 Day 0, 21, 42 IM, LNPChallenge with prME 250 μg Dengue 2/99345 s.c 4 (Construct IM, LNP (5logPFU) 21) 5 μg 5 Dengue 3 Day 0, 21, 42 IM, LNP Challenge with prME 250μg Dengue 3/16562 s.c 6 (Construct IM, LNP (5log PFU) 19) 5 μg 7 Dengue4 Day 0, 21, 42 IM, LNP Challenge with prME 250 μg Dengue 4/1036 s.c 8(Construct IM, LNP (5log PFU) 17) 5 μg 9 prME Combo Day 0, 21, 42 IM,LNP Challenge with (Post- 1000 μg Total Dengue 1/03135 s.c Formulation(250 μg of each) (5log PFU) 10 Mix) IM, LNP (Constructs 20 μg Total 15,17, 19, (5 μg of each) and 21) 11 prME Combo Day 0, 21, 42 IM, LNPChallenge with (Post- 1000 μg Total Dengue 2/99345 s.c Formulation (250μg of each) (5log PFU) 12 Mix) IM, LNP (Constructs 20 μg Total 15, 17,19, (5 μg of each) and 21) 13 prME Combo Day 0, 21, 42 IM, LNP Challengewith (Post- 1000 μg Total Dengue 3/16562 s.c Formulation (250 μg ofeach) (5log PFU) 14 Mix) IM, LNP (Constructs 20 μg Total 15, 17, 19, (5μg of each) and 21) 15 prME Combo Day 0, 21, 42 IM, LNP Challenge with(Post- 1000 μg Total Dengue 4/1036 s.c Formulation (250 μg of each)(5log PFU) 16 Mix) IM, LNP (Constructs 20 μg Total 15, 17, 19, (5 μg ofeach) and 21) 17 prME Combo Day 0, 21, 42 IM, LNP Challenge with (Post-1000 μg Total Dengue 2/99345 s.c Formulation (250 μg of each) (5log PFU)Mix) (Constructs 15, 17, 19, and 22) 18 H10N8-FLAG Day 0, 21, 42 IM, LNPChallenge with 250 μg Dengue 2/99345 s.c (5log PFU) 19 Naive — —Challenge with Dengue 1/03135 s.c (5log PFU) 20 Naive — — Challenge withDengue 2/99345 s.c (5log PFU) 21 Naive — — Challenge with Dengue 3/16562s.c (5log PFU) 22 Naive — — Challenge with Dengue 4/1036 s.c (5log PFU)Collect serum on day 20, 41, 62, and 92 for serotype-specificneutralization assay Collect serum on day 62 (pre-challenge), 63-66, 68,70, 72, 76, and 92 (end of In-life) to determine serum viral load

Example 39 Dengue 2 prME Challenge Study in AG129 Mice

The instant study was designed to evaluate the efficacy of four DENVmRNA vaccine constructs (constructs 21-24 in Table 38) in AG129 micechallenge assays. The schedule of the challenge study was shown in FIG.38A. The DENV mRNA vaccines were formulated in lipid nanoparticles(e.g., MC3 formulation) and administered to the AG129 miceintramuscularly on days 0 and 21. Dosage of the vaccines were 2 μg or 10μg per immunization. Heat inactivated D2Y98P strain was used as anegative control to vaccinate the mice. Naïve AG129 mice withoutimmunization were also used as control.

Immunized AG129 mice were challenged on day 42 post initial immunizationwith Dengue D2Y98P virus (s.c., 1e5 PFU per mouse). AG129 mice sera werecollected on days 20 and 41 post initial immunization and used forserotype-specific neutralization assays. Mice immunized with any of thefour DENV mRNA vaccine constructs survived, while the control mice died.These data demonstrate that, after lethal challenge, there was 100%protection provided by each mRNA vaccine construct, regardless of dose.The weights and health of the mice were monitored and the results wereplotted in FIGS. 38C-38D.

Mice sera collected from mice immunized with 2 μg of the DENV mRNAvaccines were able to neutralize several DENV 2 strains and variationsin the neutralization ability between the tested mRNA vaccines andbetween different DENV 2 strains were observed (FIG. 39).

Example 40 DENV prME Challenge Study in AG129 Mice Model

Shown in Table 40 is the design of a DENV prME challenge study in AG129mice, including the mRNA constructs tested, the vaccination schedule,the dosage, the challenge strains, and the serum collection schedule.

Indicated DENV mRNA vaccine encoding prME antigen epitopes, or vaccinesthereof, were used to immunize AG129 mice. The vaccines were formulatedin lipid nanoparticles (e.g., MC3 formulation) and administered to themice intramuscularly on days 0 and 21. Dosages of the vaccines were 2 μgor 10 μg per immunization. In experiments where a combination ofdifferent DENV mRNA vaccines were used, 2 μg of each mRNA vaccine wasused. Naïve AG129 mice without immunization were used as control. AG129mice sera were collected on days 20 and 41 post initial immunization andused for serotype-specific neutralization assays.

Immunized AG129 mice were challenged on day 42 post initial immunizationwith Dengue D2Y98P virus (s.c., 1e5 PFU per mouse). The weights andhealth of the mice were monitored for 14 days post infection and theresults were plotted in FIGS. 40A-40I.

TABLE 40 DENV prME Challenge Study Design in AG129 Mice Group VaccineDosage/ Serum/ n = 5 Vaccine Schedule Route PBMCs Challenge Readout  1Dengue 1 Day 0, IM, LNP, Collect serum on Challenge with Monitor prME 2110 μg day 20 and 41 for 1e5 PFU per weights (Construct 15)serotype-specific mouse of and health neutralization D2Y98P SC for 14assay injection days p.i. (Day 42)  2 Day 0, IM, LNP, 21 2 μg  3 Dengue2 Day 0, IM, LNP, prME 21 10 μg (Construct 21)  4 Day 0, IM, LNP, 21 2μg  5 Dengue 3 Day 0, IM, LNP, prME 21 10 μg  6 (Construct 19) Day 0,IM, LNP, 21 2 μg  7 Dengue 4 Day 0, IM, LNP, prME 21 10 μg  8 (Construct17) Day 0, IM, LNP, 21 2 μg  9 H2Kb Day 0, IM, LNP, Collect andMultitope 21 10 μg cryopreserve (Construct 25) PBMCs on day 20 and 41;Ship to Valera 10 Day 0, IM, LNP, 21 2 μg 11 Non-H2Kb Day 0, IM, LNP,Multitope 21 10 μg 12 (Construct 26) Day 0, IM, LNP, 21 2 μg 13 prME Day0, IM, LNP, Collect serum on Combo + 21 10 μg Total day 20 and 41 forH2Kb (2 μg of serotype-specific Multitope each) neutralization(Constructs 15, assay 17, 19, and 21) (Post7) 14 prME Day 0, IM, LNP,Combo + 21 10 μg Total non-H2Kb (2 μg of Multitope each) (Constructs 15,17, 19, 21, and 26) (Post7) 15 prME Combo Day 0, IM, LNP, (Constructs15, 21 8 μg Total 17, 19, and 21) (2 μg of (Post7) each) 16 prME Day 0,IM, LNP, Combo + 21 10 μg Total H2Kb (2 μg of Multitope each)(Constructs 15, 17, 19, 21 and 25) (Post1) 17 prME Day 0, IM, LNP,Combo + 21 10 μg Total non-H2Kb (2 μg of Multitope each) (Constructs 15,17, 19, 21, and 26) (Post1) 18 prME Combo Day 0, IM, LNP, (Constructs15, 21 8 μg Total 17, 19, and 21) (2 μg of (Post1) each) 19 Dengue 2 Day0, IM, LNP, Collect serum on prME 21 2 μg day 20 and 41 for (Construct22) Dengue 2-specific 20 Naive Day 0, Tris/Sucrose neutralization 21assay

Example 41 Virus-Like Particles

The antigens produced from the DENV prME mRNA vaccines of the presentdisclosure, when expressed, are able to assemble into virus-likeparticles (VLPs). The instant study was designed to evaluate theimmunogenicity of the VLPs by negative stain electron microscopeimaging. As shown in FIG. 41, DENV mRNA vaccine constructs 21-24 wereexpressed and VLPs were assembled an isolated. The VLPs were visualizedunder negative stain electron microscopy. Construct 23 is the vaccineconstruct used by Sanofi in its DENV vaccines. Constructs 21, 22, and 24produced more uniform VLPs, suggesting that these VLPs may be moresuperior in their immunogenicity than the VLPs produced from construct23.

Example 42 Efficacy of CHIKV mRNA Vaccine X Against CHIKV in AG129 Mice

Study Design

Chikungunya virus (CHIKV) 181/25 strain is an attenuated vaccine strainthat was developed by the US Army via multiple plaque-to-plaque passagesof the 15561 Southeast Asian human isolate (Levitt et al.). It is welltolerated in humans and is highly immunogenic. It produces small plaquesand has decreased virulence in infant mice and nonhuman primates. Whenthe attenuated virus is administered to immunodeficient AG129 mice(lacking the IFN-α/β and γ receptors) the mice succumb to a lethaldisease within 3-4 days with ruffled fur and weight loss (Partidos, etal. 2011 Vaccine).

This instant study was designed to evaluate the efficacy of CHIKVcandidate vaccines as described herein in AG129 mice (Table 41). Thestudy consisted of 14 groups of female 6-8 week old AG129 mice (Table41). Groups 1-4, 7-8, and 10-15 were vaccinated with CHIKV vaccine X viathe intramuscular (IM; 0.05 mL) route on Day 0 and select groupsreceived an additional boost on Day 28. Control Groups 9 and 16 receivedvehicle (PBS) only on Days 0 and 28 via IM route (0.05 mL). Regardlessof vaccination schedule, Groups 1-4 and 7-9 were challenged on Day 56while Groups 10-16 were challenged on Day 112 using the CHIKV 181/25strain (stock titer 3.97×10⁷ PFU/mL, challenge dose 1×10⁴ PFU/mouse).For virus challenge, all mice received a lethal dose (1×10⁴ PFU) ofChikungunya (CHIK) strain 181/25 via intradermal (ID) route (0.050 mLvia footpad). All mice were monitored for 10 days post infection forweight loss, morbidity, and mortality. Each mice was assigned a heathscore based on Table 5. Mice displaying severe illness as determinedby >30% weight loss, a health score of higher than 5, extreme lethargy,and/or paralysis were euthanized with a study endpoint of day 10 postvirus challenge. Test bleeds via retro-orbital (RO) collection wereperformed on mice from all groups on Days −3, 28, and 56. Mice fromGroups 10-16 were also bled on Days 84 & 112. Mice that survivedchallenge were also terminally bled on Day 10 post challenge. Serumsamples from mice (Days −3, 28, 56, 84, 112 and surviving mice) werekept frozen (−80° C.) and stored until they were tested for reactivityin a semi quantitative ELISA for mouse IgG against either E1, E2 orCHIKV lysate.

Experimental Procedure

Intramuscular (IM) Injection of Mice

1. Restrain the animal either manually, chemically, or with a restraintdevice.

2. Insert the needle into the muscle. Pull back slightly on the plungerof the syringe to check proper needle placement. If blood is aspirated,redirect the needle and recheck placement again.

3. Inject appropriate dose and withdraw needle. Do not exceed maximumvolume. If the required volume exceeds the maximum volume allowed,multiple sites may be used with each receiving no more than the maximumvolume.

4. The injection site may be massaged gently to disperse the injectedmaterial.

Intradermal (ID) Injections of Mice

1. Restrain the animal either manually, chemically, or with a restraintdevice.

2. Carefully clip the hair from the intended injection site. Thisprocedure can be done upon animals arriving or the day before anyprocedures or treatments are required.

3. Lumbar area is the most common site for ID injections in all species,but other areas can be used as well.

4. Pinch or stretch the skin between your fingers (or tweezers) toisolate the injection site.

5. With the beveled edge facing up, insert the needle just under thesurface between the layers of skin. Inject the appropriate dose andwithdraw needle. A small bleb will form when an ID injection is givenproperly.

6. If the required volume exceeds the maximum volume allowed, multiplesites may be used with each receiving no more than the maximum volume.

Retro-orbital Bleeding in Mice

1. Place the mice in the anesthesia chamber and open oxygen line and setto 2.5% purge. Start flow of anesthesia at 5% isoflurane.

2. Once the animal becomes sedate, turn anesthesia to 2.5%-3% isofluraneand continue to expose the animal to the anesthesia. Monitor the animalto avoid breathing becoming slow.

3. Remove the small rodent from anesthesia chamber and place on its backwhile restraining with left hand and scruff the back of the animal'sneck, so it is easy to restrain and manipulate while performing theprocedure with the right hand.

4. With a small motion movement, place the capillary tube in the cornerof the animal's eye close to the nostril, and rotate or spin theHematocrit glass pipette until blood start flowing out. Collect theappropriate amount of blood needed into the appropriate labeled vial.

5. Monitor the animal after retro-orbital bleeding is done for at least10-15 seconds to ensure hemostasis.

6. Place the animal back to its original cage and monitor for any otherproblems or issues caused while manipulating animal due to theprocedure.

Observation of Mice

1. Mice were observed through 10 days post infection (11 days total,0-10 days post infection).

2. Mice were weighed daily on an Ohause scale and the weights arerecorded.

3. Survival and health of each mouse were evaluated once time a dayusing a scoring system of 1-7 described in Table 5.

Infection

On either Day 56 (Groups 1-4, 7-9) or Day 112 (Groups 10-16) groups of 5female 6-8 week old AG129 mice were infected via intradermal injectionwith 1×10⁴ PFU/mouse of the 181/25 strain of Chikungunya diluted in PBS.The total inoculation volume was 0.05 mL administered in the rearfootpad of each animal. Mice were anesthetized lightly using 2-5% v/v ofisoflurane at ˜2.5 L/min of 02 (VetEquip IMPAC6) immediately prior toinfection.

Dose Administration

In this study mice were administered 0.04 μg, 2 μg, or 10 μg of variousformulations of the CHIKV vaccine X or vehicle alone (PBS) on either Day0 or on Days 0 and 28 via the intramuscular route (0.05 mL). Thematerial was pre-formulated by the Client and diluted in PBS by IBTprior to dosing as per instructions provided by the Client.

Results

Mice were immunized once (Day 0) or twice (Days 0 & 28) with either 0.04μg, 2 μg, or 10 μg of Chikungunya vaccine X and were challenged withCHIKV strain 181/25 on either Day 56 (Groups 1-4, 7-9) or on Day 112(Groups 10-16). Mice were monitored for a total of 10 days postinfection for health and weight changes. Mice that received either 2 μgor 10 μg of the CHIKV vaccine X either once (Day 0) or twice (Days 0 and28) were fully protected (100%) regardless of whether the mice werechallenged 56 days or 112 days after the initial vaccination (FIGS.42A-42B, Table 44). Mice receiving 0.04 μg of the CHIKV vaccine were notprotected at all from lethal CHIKV infection. This efficacy data issupported by the health scores observed in the vaccinated mice in thatthe protected mice displayed little to no adverse health effects of aCHIKV infection (FIGS. 44A-44B). Weight loss is not a strong indicatorof disease progression in the CHIKV AG129 mouse model (FIGS. 43A-43B).

Mice immunized with the CHIKV vaccine X showed increased antibody titersagainst CHIKV E1, E2 and CHIKV lysate as compared to the vehicle only(PBS) treated groups. Serum binding against the virus lysate yielded thehighest antibody titers for all vaccinated groups (FIGS. 45A-45C,46A-46C, 47A-47C, 48A-48C). Overall, the antibody titers were dosedependent with the highest titers observed in serum from mice vaccinatedwith 10 μg of CHIKV vaccine X while the lowest titers were observed inserum from mice vaccinated with 0.04 μg of the CHIKV vaccine X.Similarly, higher titers were observed in serum from mice vaccinatedtwice (Days 0 and 28) as compared to serum from mice vaccinated onlyonce (Day 0). Serum obtained on Day 112 post initial vaccination stillyielded increased antibody titers in mice that received either 10 μg or2 μg of CHIKV vaccine X (FIGS. 47A-47C).

Serum from mice groups 10-16, 112 days post immunization were alsotested in a Plaque Reduction Neutralization Test (PRNT). Serum from eachmice was diluted from 1/20 to 1/40960 and assessed for its ability toreduce CHIKV plaque formation. The results were shown in Table 46.

TABLE 41 CHIKV Challenge Study Design in AG129 mice Dose Group* (IM (n =5) Vaccine Schedule route) Challenge Bleeds 1 VAL- Day 0 10 μg Challengewith Pre-bleed for 2 181388 Day 0 & 28 1 × 10⁴ PFU per serum via RO 3Day 0 2 μg mouse of CHIK route on days −3, 4 Day 0 & 28 181/25 via ID28, 56, (all injection groups) & 84, 112 on day 56. (groups 10-16 7 Day0 0-4 μg Weights and only). 8 Day 0 & 28 health for 10 Terminal bleed 9PBS Day 0 & 28 — days following surviving mice on infection. day 10 post10 VAL- Day 0 10 μg Challenge with challenge. 11 181388 Day 0 & 28 1 ×10⁴ PFU per Serum stored 12 Day 0 2 μg mouse of CHIK at −80° C. 13 Day 0& 28 181/25 via ID 14 Day 0 0-4 μg injection on day 15 Day 0 & 28 112.Weights and 16 PBS Day 0 & 28 — health for 10 days following infection.*No group 5 or 6 in this study

TABLE 42 Equipment and Software Item Vendor Cat#/Model Syringes BDVarious Animal Housing InnoVive Various Scale Ohause AV2101 Prismsoftware GraphPad N/A Microplate Washer BioTek ELx405 Plate reader withSoftMax Pro Molecular Devices VersaMax version 5.4.5

TABLE 43 ELISA Reagents Storage Name Supplier cat# Temperature NotesDPBS 1X, sterile Corning 21-031- Ambient For dilution of coating antigenCM or equivalent StartingBlock T20 Thermo Scientific 2-8° C. Forblocking non-specific (PBS) Blocking 37539 binding and use as diluent ofBuffer Standards, unknown test sera and detection antibody SureBlueReserve KPL 53-00-02 or 2-8° C. N/A TMB Microwell equivalent PeroxidaseSubstrate (1- Component) DPBS powder, non- Corning 55-031-PB 2-8° C. Usedeionized water to sterile or equivalent dissolved DPBS powder from onebottle to a final volume of 10 liters of 1X DPBS TWEEN-20 Sigma-AldrichAmbient Add 5 mL TWEEN-20 to 10 P1379-500ML or liters of 1X DPBS and mixequivalent well to prepare DPBS + 0.05% TWEEN-20 Wash Buffer forautomatic plate washer

TABLE 44 ELISA Reagents Critical Reagent Please note: Coating antigensand standards Supplier cat# Storage are stored as single-use aliquots.and/or lot# Temperature Assay Parameter Coating antigens CHIKVrecombinant E1 glycoprotein, IBT Bioservices, −70° C. or below 400ng/well expressed in 293 mammalian cells IBT's lot 08.11.2015 BCA =0.351 mg/mL CHIKV recombinant E2 glycoprotein, ImmunoDx, cat# −70° C. orbelow 400 ng/well expressed in E. coli IBT's BCA = 1.291 80002, lotmg/mL 10MY4 CHIKV 181/25 lysate from sucrose- IBT Bioservices, −70° C.or below 300 ng/well purified viruses, lysed by sonication lot11.23.2015 IBT's BCA = 1.316 mg/mL Standards Anti-E1 positive controlPooled mouse IBT Bioservices −70° C. or below Assigned, 30,812 serumfrom survivors of BS-1842 group Antibody Units/mL 4 (vaccinated with E1mRNA 10 μg, ID, against E1 protein LNP on study days 0 and 28) day 66terminal bleeds (10 days after CHIKV infection) Anti-E2 positivecontrol, Pooled mouse IBT Bioservices −70° C. or below Assigned, 16912serum from survivors of BS-1842 group Antibody Units/mL 8 (vaccinatedwith E2 mRNA 10 μg, ID, against E2 protein LNP on study days 0 and 28)day 66 Assigned 14,200 terminal bleeds (10 days after CHIKV AntibodyUnits/mL infection) Detection antibody Anti-mouse IgG (H + L)-HRP KPL,cat# 474- 2-8° C. 1:6000 dilution 1806, lot 140081

TABLE 45 Survival Percentage a. Groups 1-4 and 7-9, Day 56 Challenge 10μg 2 μg 0.4 μg Days 10 μg Day 0 & 2 μg Day 0 0.4 μg Day 0 & p.i. Day 028 Day 0 & 28 Day 0 28 PBS  0 100 100 100 100 100 100 100  3 80  4 0 4080  5 0 0 10 100 100 100 100 b. Groups 10-16, Day 112 Challenge 10 μg 2μg 0.04 μg Days 10 μg Day 0 & 2 μg Day 0 0.04 μg Day 0 & p.i. Day 0 28Day 0 & 28 Day 0 28 PBS  0 100 100 100 100 100 100 100  3 80 80  4 20 2050  5 0 0 0 10 100 100 100 100

TABLE 46 CHIKV Plaque Reduction Neutralization Test (PRNT) Serumdilutions from 1/20 to 1/40960 Expt info Vaccination CHIKV strain samplePRNT80 PRNT50 GP# regimen 37997 ID titer titer 10 Day 0, CHIKV 1  1/160 1/640 IM/10 μg 37997 2  1/320  1/320 working stock 3  1/160  1/640titer = 4  1/160    1/1280 780 PFU/ml 5  1/320    1/1280 11 Day 0/Day28, 1  1/640    1/2560 IM/10 μg 2    1/1280    1/1280 3  1/320    1/25604  1/640    1/5120 5    1/1280    1/5120 12 Day 0, 1  1/20  1/80 IM/2 μg2  1/40  1/320 3 <1/20  1/160 PRNT80 4 <1/20  1/160 cutoff 5 <1/20  1/2013 Day 0, 8 PFU 1  1/80  1/320 Day 28, 2  1/80  1/640 IM/2 μg 3  1/20 1/320 4  1/20  1/320 5  1/320  1/640 14 Day 0, 1 <1/20 80 IM/0.4 μg 2<1/20 <1/20 3 <1/20 <1/20 4 <1/20 <1/20 5 <1/20 <1/20 15 Day 0, PRNT50 1<1/20 <1/20 Day 28, cutoff 2 <1/20 80 IM/0.4 μg 20 PFU 3 <1/20 <1/20 4<1/20 <1/20 5 <1/20 <1/20 16 Vehicle 1 <1/20 <1/20 Day 0/Day 28 2 <1/20<1/20 3 <1/20 <1/20 4 <1/20 <1/20 5 <1/20 <1/20

Example 43 Immunogenicity of Chikungunya Polyprotein (C-E3-E2-6K-E1)mRNA Vaccine Candidate in Rats

Sprague Dawley rats (n=5) were vaccinated with 20 μg of MC-3-LNPformulated mRNA 30 encoded CHIKV polyprotein (C-E3-E2-6K-E1) (SEQ ID NO:13). The rats were vaccinated on either Day 0 or Days 0 and 14 or Days0, 14 and 28 via IM delivery. Sera was collected on days −3, 14, 28 and42 for ELISA testing. FIG. 58 demonstrated that there was at least a twolog increase in antibody titer against CHIKV lysate post 3rd vaccinationwith the mRNA vaccine in normal rats.

Example 44 Evaluation of T Cell Activation of Chikungunya P 5Polyprotein (C-E3-E2-6K-E1) mRNA Vaccine Candidate

C57BL/6 mice (n=6 experimental group; n=3 control group) were vaccinatedwith 10 μg of MC-3-LNP formulated mRNA encoded CHIKV polyprotein(C-E3-E2-6K-E1) (SEQ ID NO: 13). The mice were vaccinated on either Day0 or Days 0 and 28 (boost) via IM delivery. Sera was collected on days3, 28 and 42 for ELISA testing. Animals were sacrificed on day 42 andspleens were harvested for immunological evaluation of T cells. Spleniccells were isolated and analyzed by FACS. Briefly, spleens were removed,cells isolated, and stimulated in vitro with immunogenic peptides foundwithin either C, E1, or E2 region of CHIKV that are known to be CD8epitopes in B6 mice. The readout for this assay was cytokine secretion(IFN-gamma and TNF-alpha), which reveals whether the vaccine inducedantigen-specific T cell responses. No CD8 T cell responses were detectedusing the E2 or C peptide (baseline levels of IFN-gamma and TNF-alpha),whereas there was a response to the E1-corresponding peptide (average ofabout 0.4% IFN-gamma and 0.1% TNF). The peptides were used to stimulateT cells used in the study were E1=HSMTNAVTI (SEQ ID NO: 300),E2=IILYYYELY (SEQ ID NO: 301), and C=ACLVGDKVM (SEQ ID NO: 302).

FIG. 59 shows that the polyprotein-encoding CHIKV polyprotein vaccineelicited high antibody titers against the CHIKV glycoproteins. FIGS. 60and 61A-61B show T cell activation by E1 peptide.

EQUIVALENTS

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A Zika virus (ZIKV) immunogenic composition,comprising: a messenger ribonucleic acid (mRNA) polynucleotide having anopen reading frame encoding a ZIKV antigenic polypeptide formulated in alipid nanoparticle.
 2. The ZIKV immunogenic composition of claim 1,wherein the antigenic polypeptide is a ZIKV polyprotein.
 3. The ZIKVimmunogenic composition of claim 1, wherein the antigenic polypeptide isselected from the group consisting of a ZIKV capsid protein, a ZIKVpremembrane/membrane protein, a ZIKV envelope protein, a ZIKVnon-structural protein 1, a ZIKV non-structural protein 2A, a ZIKVnon-structural protein 2B, a ZIKV non-structural protein 3, a ZIKVnon-structural protein 4A, a ZIKV non-structural protein 4B, and a ZIKVnon-structural protein
 5. 4. The ZIKV immunogenic composition of claim3, wherein the antigenic polypeptide comprises a ZIKV capsid protein, aZIKV premembrane/membrane protein, and a ZIKV envelope protein.
 5. TheZIKV immunogenic composition of claim 3, wherein the antigenicpolypeptide comprises a ZIKV capsid protein and a ZIKVpremembrane/membrane protein.
 6. The ZIKV immunogenic composition ofclaim 3, wherein the antigenic polypeptide comprises a ZIKV capsidprotein and a ZIKV envelope protein.
 7. The ZIKV immunogenic compositionof claim 3, wherein the antigenic polypeptide comprises a ZIKVpremembrane/membrane protein and a ZIKV envelope protein.
 8. The ZIKVimmunogenic composition of claim 1, wherein the open reading frameencodes at least two ZIKV antigenic polypeptides.
 9. The ZIKVimmunogenic composition of claim 1, wherein the immunogenic compositioncomprises at least two RNA polynucleotides each having an open readingframe encoding a ZIKV antigenic polypeptide.
 10. The ZIKV immunogeniccomposition of claim 1, wherein the antigenic polypeptide is obtainedfrom ZIKV strain MR 766, ZIKV strain ACD75819 or ZIKV strain SPH2015.11. The ZIKV immunogenic composition of claim 1, wherein the RNApolynucleotide comprises a chemical modification.
 12. The ZIKVimmunogenic composition of claim 11, wherein the chemical modificationis a N1-methylpseudouridine.
 13. The ZIKV immunogenic composition ofclaim 11, wherein at least 80% of the uracil in the open reading framehave a chemical modification.
 14. The ZIKV immunogenic composition ofclaim 13, wherein 100% of the uracil in the open reading frame has achemical modification.
 15. The ZIKV immunogenic composition of claim 1,wherein the RNA polynucleotide further encodes a 5′ terminal cap. 16.The ZIKV immunogenic composition of claim 15, wherein the 5′ terminalcap is 7 mG(5′)ppp(5′)NlmpNp.
 17. The ZIKV immunogenic composition ofclaim 1, wherein the lipid nanoparticle comprises a cationic lipid, aPEG-modified lipid, a sterol and a non-cationic lipid.
 18. The ZIKVimmunogenic composition of claim 17, wherein the lipid nanoparticlecomprises a molar ratio of about 20-60% cationic lipid, 0.5-15%PEG-modified lipid, 25-55% sterol, and 5-25% non-cationic lipid.
 19. TheZIKV immunogenic composition of claim 17, wherein the cationic lipid isan ionizable cationic lipid, the non-cationic lipid is a neutral lipid,and the sterol is a cholesterol.
 20. The ZIKV immunogenic composition ofclaim 17, wherein the cationic lipid is selected from the groupconsisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate.
 21. A method ofinducing an immune response in a subject, the method comprisingadministering to the subject a ZIKV immunogenic composition comprising amessenger ribonucleic acid (mRNA) polynucleotide having an open readingframe encoding a ZIKV antigenic polypeptide, in an amount effective toproduce an antigen-specific immune response in the subject.
 22. A Zikavirus (ZIKV) immunogenic composition, comprising a ribonucleic acid(RNA) polynucleotide having an open reading frame encoding a ZIKVantigenic polypeptide, a 5′ terminal cap, and a chemical modification,wherein the immunogenic composition is formulated within a lipidnanoparticle.
 23. The ZIKV immunogenic composition of claim 22, whereinat least 80% of the uracil in the open reading frame have a chemicalmodification.
 24. A Zika virus (ZIKV) immunogenic composition,comprising a messenger ribonucleic acid (mRNA) polynucleotide having anopen reading frame encoding a ZIKV polypeptide linked to a signalpeptide.
 25. The ZIKV immunogenic composition of claim 24, wherein theZIKV antigenic polypeptide is a ZIKV PrM protein, a ZIKV envelope (E)protein, or a ZIKV prME fusion protein.
 26. The ZIKV immunogeniccomposition of claim 24, wherein the ZIKV antigenic polypeptide is fusedto a signal peptide selected from the group consisting of: a HuIgGksignal peptide (METPAQLLFLLLLWLPDTTG; SEQ ID NO: 125); IgE heavy chainepsilon-1signal peptide (MDWTWILFLVAAATRVHS; SEQ ID NO: 126); Japaneseencephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS; SEQ ID NO:128) and VSVg protein signal sequence (MKCLLYLAFLFIGVNCA; SEQ ID NO:131).
 27. The immunogenic composition of claim 1, further comprising aRNA polynucleotide having an open reading frame encoding a CHIKVantigenic polypeptide and/or a DENV antigenic polypeptide.